Compositions And Methods For Inhibiting Expression Of CD45 Gene

ABSTRACT

The invention relates to a double-stranded ribonucleic acid (dsRNA) for inhibiting the expression of the CD45 gene.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.13/612,521 filed Sep. 12, 2012, (allowed), which is a continuation ofU.S. application Ser. No. 12/867,230, now U.S. Pat. No. 8,288,525,issued Oct. 16, 2012, which is the National Stage of InternationalApplication No. PCT/US2009/033931, filed Feb. 12, 2009, all which claimthe benefit of U.S. Provisional Application No. 61/028,162, filed Feb.12, 2008. The entire contents of these applications are herebyincorporated by reference in the present application.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted via EFS-Web and is hereby incorporated by reference in itsentirety. Said ASCII copy, created on Nov. 7, 2014, is named28122US_CRF_sequencelisting.txt and is 126,484 bytes in size.

GOVERNMENT SUPPORT

This invention was made with government support under HDTRA1-07-C-0082awarded by the Defense/Defense Threat Reduction Agency, andHHSN266200600012C awarded by the Department of Health and HumanServices/NIH/NAIAD. The government has certain rights in the invention.

FIELD OF THE INVENTION

This invention relates to double-stranded ribonucleic acid (dsRNA), andits use in mediating RNA interference to inhibit the expression of theCD45 gene and the use of the dsRNA to treat infectious diseases andautoimmune disease.

BACKGROUND OF THE INVENTION

CD45 is a hematopoietic cell-specific transmembrane protein tyrosinephosphatase essential for T and B cell antigen receptor-mediatedsignaling and also plays a important role in cytokine receptorsignaling, chemokine and cytokine response and apoptosis regulation inmultiple different leukocyte cell subsets (T cells, B cells, NK cells,myeloid cells, granulocytes, and dendritic cells). CD45 constitutesnearly 10% of T and B cell surface protein. The protein includes a largeextracellular domain, and a phosphatase containing cytosolic domain.CD45 may act as both a positive and negative regulator depending on thenature of the stimulus and the cell type involved. CD45 RNA transcriptsare alternatively spliced at the N-terminus, which results inextracellular domains of various sizes. The protein controls theactivity of Src-family kinases, which if left unregulated, can causecancer and autoimmunity. Mice and humans lacking CD45 expression havebeen shown to be immunodeficient.

Multiple human or rodent mutations that result in altered CD45expression or functional activity are associated with distinctmalignancies, including autoimmunity, immunodeficiency, overt activationof T cells, susceptibility to infection, type I or type II associatedimmune disorders, and haemotologic malignancies (reviewed in Tchilianand Beverly, Trends in Immunology, 2006).

Double-stranded RNA molecules (dsRNA) have been shown to block geneexpression in a highly conserved regulatory mechanism known as RNAinterference (RNAi). WO 99/32619 (Fire et al.) discloses the use of adsRNA of at least 25 nucleotides in length to inhibit the expression ofthe unc-22 gene in C. elegans. dsRNA has also been shown to degradetarget RNA in other organisms, including plants (see, e.g., WO 99/53050,Waterhouse et al.; and WO 99/61631, Heifetz et al.), Drosophila (see,e.g., Yang, D., et al., Curr. Biol. (2000) 10:1191-1200), and mammals(see WO 00/44895, Limmer; and DE 101 00 586.5, Kreutzer et al.).

SUMMARY OF THE INVENTION

The invention provides double-stranded ribonucleic acid (dsRNA), as wellas compositions and methods for inhibiting the expression of the CD45gene in a cell or mammal using such dsRNA. The invention also providescompositions and methods for treating pathological conditions anddiseases caused by the expression of the CD45 gene, such as infectiousdisease and autoimmune disease. The dsRNA featured in the inventionincludes an RNA strand (the antisense strand) having a region which isless than 30 nucleotides in length, generally 19-24 nucleotides inlength, and which is substantially complementary or fully complementaryto the corresponding region of an mRNA transcript of the CD45 gene.

In one aspect, the invention features, double-stranded ribonucleic acid(dsRNA) molecules for inhibiting the expression of the CD45 gene. ThedsRNA includes at least two sequences that are complementary, e.g.,substantially or fully complementary, to each other. The dsRNA includesa sense strand including a first sequence and an antisense strandincluding a second sequence. The antisense strand includes a nucleotidesequence which is substantially or fully complementary to thecorresponding region of an mRNA encoding CD45, and the region ofcomplementarity is less than 30 nucleotides in length, generally 19-24nucleotides in length, e.g., 19 to 21 nucleotides in length. In someembodiments, the dsRNA is from about 10 to about 15 nucleotides, and inother embodiments the dsRNA is from about 25 to about 30 nucleotides inlength. In another embodiment, the dsRNA is at least 15 nucleotides inlength. The dsRNA, upon contacting with a cell expressing the CD45,e.g., in an assay described herein, e.g., in a P388D1 cell assay asdescribed herein (or an assay based on a cell with similar properties),inhibits the expression of the CD45 gene by at least 20% or 25%, andpreferably by at least 35%, or preferably by at least 40%. In oneembodiment, the CD45 dsRNA is formulated in a stable nucleic acidparticle (SNALP).

The dsRNA molecules featured in the invention include dsRNAs that cleavea CD45 mRNA in a target sequence selected from the group consisting ofSEQ ID NOs:97-144. The dsRNAs featured herein also include dsRNAs havinga first sequence selected from the group consisting of the sensesequences of Tables 2, 4 and 5, and a second sequence selected from thegroup consisting of the antisense sequences of Tables 2, 4 and 5. ThedsRNA molecules featured in the invention can include naturallyoccurring nucleotides or can included at least one modified nucleotide,such as a 2′-O-methyl modified nucleotide, a nucleotide including a5′-phosphorothioate group, and a terminal nucleotide linked to acholesteryl derivative or dodecanoic acid bisdecylamide group.Alternatively, the modified nucleotide may be chosen from the group of:a 2′-deoxy-2′-fluoro modified nucleotide, a 2′-deoxy-modifiednucleotide, a locked nucleotide, an abasic nucleotide, 2′-amino-modifiednucleotide, 2′-alkyl-modified nucleotide, morpholino nucleotide, aphosphoramidate, and a non-natural base comprising nucleotide.Generally, the first sequence of the dsRNA is selected from the groupconsisting of the sense sequences of Tables 2, 4 and 5, and the secondsequence is selected from the group consisting of the antisensesequences of Tables 2, 4 and 5.

In another aspect, the invention provides a cell including a dsRNAtargeting CD45. The cell can be a mammalian cell, such as a human cell.

In another aspect, the invention features a pharmaceutical compositioncontaining a dsRNA, such as a dsRNA described herein, e.g., in Tables 2,4 and 5, and a pharmaceutically acceptable carrier. In one embodiment,the pharmaceutical composition does not include another agent thatsilences gene expression. In another embodiment, the pharmaceuticalcomposition does not include another dsRNA, e.g., a dsRNA of a length oroverhang structure described herein. In another embodiment, thepharmaceutical composition consists of or consists essentially of thesubject dsRNA. In another embodiment, the pharmaceutical compositionincludes more than one dsRNA. In yet other embodiments, thepharmaceutical composition includes more than one but not more than 2, 3or 4 dsRNAs.

In another aspect, the invention provides a method for inhibiting theexpression of the CD45 gene in a cell, including the following steps:

-   -   (a) introducing into the cell a double-stranded ribonucleic acid        (dsRNA), e.g., a dsRNA described herein, e.g., a dsRNA that        cleaves a CD45 mRNA in a target sequence selected from the group        consisting of SEQ ID NOs:97-144, wherein the dsRNA includes at        least two sequences that are complementary, e.g., substantially        or fully complementary, to each other. The dsRNA includes a        sense strand including a first sequence and an antisense strand        including a second sequence. The antisense strand includes a        region of complementarity which is substantially or fully        complementary to the corresponding region of an mRNA encoding        CD45, and wherein the region of complementarity is less than 30        nucleotides in length, generally 19-24 nucleotides in length,        and preferably, wherein the dsRNA, upon contact with a cell        expressing the CD45, inhibits expression of the CD45 gene by at        least 20%, at least 25%, or at least 40%; and    -   (b) maintaining the cell produced in step (a) for a time        sufficient to obtain degradation of the mRNA transcript of the        CD45 gene, thereby inhibiting expression of the CD45 gene in the        cell.

In another aspect, the invention provides methods for treating,preventing or managing infectious disease by administering to a patientin need of such treatment, prevention or management a therapeutically orprophylactically effective amount of one or more of the CD45 dsRNAsfeatured in the invention.

In another aspect, the invention provides methods for treating,preventing or managing autoimmune disease, including administering to apatient in need of such treatment, prevention or management atherapeutically or prophylactically effective amount of one or more ofthe CD45 dsRNAs featured in the invention.

In another aspect, the invention provides methods for treating,preventing or managing inflammation, including administering to apatient in need of such treatment, prevention or management atherapeutically or prophylactically effective amount of one or more ofthe CD45 dsRNAs featured in the invention.

In another aspect, the invention provides methods for treating,preventing or managing a viral infection, including administering to apatient in need of such treatment, prevention or management atherapeutically or prophylactically effective amount of one or more ofthe CD45 dsRNAs featured in the invention.

In another aspect, the invention provides vectors for inhibiting theexpression of the CD45 gene in a cell, including a regulatory sequenceoperably linked to a nucleotide sequence that encodes at least onestrand of one of the dsRNAs featured in the invention.

In another aspect, the invention provides a cell including a vector forinhibiting the expression of the CD45 gene in a cell. The vectorincludes a regulatory sequence operably linked to a nucleotide sequencethat encodes at least one strand of one of the CD45 dsRNAs featured inthe invention.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A and 1B are graphs showing that a lipid-formulated dsRNAtargeting CD45 delivered to mice intraperitoneally inhibited proteinexpression of CD45 in vivo in peritoneal macrophages as compared to anirreleveant similarly formulated dsRNA targeting GFP. FIG. 1Ademonstrates reduction in CD45 protein expression following a singleinjection with 10 mg/kg formulated CD45 siRNA as compared to saline orirrelevant siRNA. FIG. 1B shows two independent dose responseexperiments demonstrating that substantial reduction in CD45 expressionis seen in vivo following a single injection of 0.6-15 mg/kg formulatedCD45 siRNA relative to irrelevant siRNA.

FIG. 2 is a bar graph showing in vitro RNAi-mediated silencing of CD45in primary mouse bone-marrow derived monocytes using a lipodoidformulation.

FIG. 3 is a graph showing that genetically modified mice expressingdifferent levels of CD45 are protected from 30,000×LD50 Ebola-Zairevirus challenge.

FIG. 4 is a bar graph showing in vitro RNAi-mediated silencing of CD45in KG1, a human leukemia cell line using a lipodoid formulation.

FIGS. 5A and 5B illustrate the sequence of human CD45 cDNA (SEQ ID NO:339) as recorded at GenBank Accession No. NM_(—)002838.2 (version datedJan. 13, 2008).

FIGS. 6A and 6B illustrate the sequence of mouse CD45 cDNA (SEQ ID NO:340) as recorded at GenBank Accession No. NM_(—)011210 (version datedJan. 27, 2008).

FIGS. 7A and 7B illustrate the sequence of rhesus CD45 cDNA (SEQ ID NO:341) as recorded at GenBank Accession No. XR_(—)012672.1 (version datedJun. 14, 2006).

DETAILED DESCRIPTION OF THE INVENTION

The invention provides double-stranded ribonucleic acid (dsRNA), as wellas compositions and methods for inhibiting the expression of the CD45gene in a cell or mammal using the dsRNA. The invention also providescompositions and methods for treating pathological conditions anddiseases in a mammal caused by the expression of the CD45 gene usingdsRNA. dsRNA directs the sequence-specific degradation of mRNA through aprocess known as RNA interference (RNAi). The process occurs in a widevariety of organisms, including mammals and other vertebrates.

The dsRNAs featured in the invention includes an RNA strand (theantisense strand) having a region which is less than 30 nucleotides inlength, generally 19-24 nucleotides in length, and is substantially orfully complementary to at least part of an mRNA transcript of the CD45gene. The use of these dsRNAs enables the targeted degradation of mRNAsof genes that are implicated in autoimmunity and infectious disease inmammals. Using cell-based and animal assays, the present inventors havedemonstrated that very low dosages of these dsRNA can specifically andefficiently mediate RNAi, resulting in significant inhibition ofexpression of the CD45 gene. Thus, the methods and compositions featuredin the invention including these dsRNAs are useful for treatingautoimmunity and infectious disease.

The following detailed description discloses how to make and use thedsRNA and compositions containing dsRNA to inhibit the expression of atarget CD45 gene, as well as compositions and methods for treatingdiseases and disorders caused by the expression of CD45, such as aninfectious disease or autoimmune disease. The pharmaceuticalcompositions featured in the invention include a dsRNA having anantisense strand having a region of complementarity which is less than30 nucleotides in length, generally 19-24 nucleotides in length, and issubstantially complementary to at least part of an RNA transcript of theCD45 gene, together with a pharmaceutically acceptable carrier.

Accordingly, certain aspects featured in the invention providepharmaceutical compositions including a dsRNA targeting CD45 togetherwith a pharmaceutically acceptable carrier, methods of using thecompositions to inhibit expression of the CD45 gene, and methods ofusing the pharmaceutical compositions to treat diseases caused byexpression of the CD45 gene.

I. DEFINITIONS

For convenience, the meaning of certain terms and phrases used in thespecification, examples, and appended claims, are provided below. Ifthere is an apparent discrepancy between the usage of a term in otherparts of this specification and its definition provided in this section,the definition in this section shall prevail.

“G,” “C,” “A,” “T” and “U” each generally stand for a nucleotide thatcontains guanine, cytosine, adenine, thymidine and uracil as a base,respectively. However, it will be understood that the term“ribonucleotide” or “nucleotide” can also refer to a modifiednucleotide, as further detailed below, or a surrogate replacementmoiety. The skilled person is well aware that guanine, cytosine,adenine, thymidine and uracil may be replaced by other moieties withoutsubstantially altering the base pairing properties of an oligonucleotideincluding a nucleotide bearing such replacement moiety. For example,without limitation, a nucleotide including inosine as its base may basepair with nucleotides containing adenine, cytosine, or uracil. Hence,nucleotides containing uracil, guanine, or adenine may be replaced inthe dsRNAs featured in the invention by a nucleotide containing, forexample, inosine. In another example, adenine and cytosine anywhere inthe oligonucleotide can be replaced with guanine and uracil,respectively to form G-U Wobble base pairing with the target mRNA.Sequences including such replacement moieties are embodiments featuredin the invention.

By “CD45” as used herein is meant a CD45 mRNA, protein, peptide, orpolypeptide. The term “CD45” is also known in the art as PTPRC (proteintyrosine phosphatase, receptor type, C), B220, GP180, LCA, LY5, andT200. The sequence of human CD45 cDNA is recorded at GenBank AccessionNo. NM_(—)002838.2 (version dated Jan. 13, 2008) (see FIGS. 5A and 5B).Other human CD45 sequences are recorded at GenBank Accession Nos.NM_(—)080921.2, NM_(—)080922.2, NM_(—)080923.2, Y00062.1, Y00638.1,BC014239.2, BC017863.1, BC031525.1, BC121086.1, BC121087.1, BC127656.1,BC127657.1, AY429565.1, AY567999.1, AK130573.1, DA670254.1, DA948670.1,AY429566.1, and CR621867.1. Mouse CD45 mRNA sequences are found atGenBank Accession Nos. NM_(—)011210.2, AK054056.1, AK088215.1,AK154893.1, AK171802.1, BC028512.1, EF101553.1, L36091.1, M11934.1,M14342.1, M14343.1, M15174.1, M17320.1, and M92933.1. Rhesus monkey CD45mRNA sequence are found at GenBank Accession No. XR_(—)012672.1.

As used herein, “target sequence” refers to a contiguous portion of thenucleotide sequence of an mRNA molecule formed during the transcriptionof the CD45 gene, including mRNA that is a product of RNA processing ofa primary transcription product.

As used herein, the term “strand including a sequence” refers to anoligonucleotide including a chain of nucleotides that is described bythe sequence referred to using the standard nucleotide nomenclature.

The terms “complementary”, “fully complementary” and “substantiallycomplementary” herein may be used with respect to the base matchingbetween the sense strand and the antisense strand of a dsRNA, or betweenthe antisense strand of a dsRNA and a target sequence, as will beunderstood from the context of their use.

As used herein, and unless otherwise indicated, the term“complementary,” when used to describe a first nucleotide sequence inrelation to a second nucleotide sequence, refers to the ability of anoligonucleotide or polynucleotide including the first nucleotidesequence to hybridize and form a duplex structure under certainconditions with an oligonucleotide or polynucleotide including thesecond nucleotide sequence, as will be understood by the skilled person.Complimentary includes both fully complimentary and substantiallycomplimentary states. Fully complimentary means comlimentarity at eachnucleotide pair of to compared sequences, e.g., an antisence strand andthe corresponding portion of a target mRNA. For substantialcomplementarity, such conditions can, for example, be stringentconditions, where stringent conditions may include: 400 mM NaCl, 40 mMPIPES pH 6.4, 1 mM EDTA, 50° C. or 70° C. for 12-16 hours followed bywashing. Other conditions, such as physiologically relevant conditionsas may be encountered inside an organism, can apply. The skilled personwill be able to determine the set of conditions most appropriate for atest of complementarity of two sequences in accordance with the ultimateapplication of the hybridized nucleotides. In other embodiments,substantial complimentarity can mean not more than 4, 3 or 2 mismatchedbase pairs upon hybridization, while retaining the ability to hybridizeunder the conditions most relevant to their ultimate application. Wheretwo oligonucleotides are designed to form, upon hybridization, one ormore single stranded overhangs, such overhangs shall not be regarded asmismatches with regard to the determination of complementarity. Forexample, a dsRNA including one oligonucleotide 21 nucleotides in lengthand another oligonucleotide 23 nucleotides in length, wherein the longeroligonucleotide includes a sequence of 21 nucleotides that is fullycomplementary to the shorter oligonucleotide, may yet be referred to as“fully complementary” for the purposes of the invention.

“Complementary” sequences, as used herein, may also include, or beformed entirely from, non-Watson-Crick base pairs and/or base pairsformed from non-natural and modified nucleotides, in as far as the aboverequirements with respect to their ability to hybridize are fulfilled.Such non-Watson-Crick base pairs includes, but not limited to, G:UWobble or Hoogstein base pairing.

As used herein, a polynucleotide which is “substantially complementaryto at least part of” a messenger RNA (mRNA) refers to a polynucleotidewhich is substantially complementary to a contiguous portion of the mRNAof interest (e.g., encoding CD45). For example, a polynucleotide iscomplementary to at least a part of a CD45 mRNA if the sequence issubstantially complementary to a non-interrupted portion of an mRNAencoding CD45.

The term “double-stranded RNA” or “dsRNA”, as used herein, refers to aribonucleic acid molecule, or complex of ribonucleic acid molecules,having a duplex structure including two anti-parallel and substantiallycomplementary, as defined above, nucleic acid strands. The two strandsforming the duplex structure may be different portions of one larger RNAmolecule, or they may be separate RNA molecules. Where the two strandsare part of one larger molecule, and therefore are connected by anuninterrupted chain of nucleotides between the 3′-end of one strand andthe 5′ end of the respective other strand forming the duplex structure,the connecting RNA chain is referred to as a “hairpin loop”. Where thetwo strands are connected covalently by means other than anuninterrupted chain of nucleotides between the 3′-end of one strand andthe 5′ end of the respective other strand forming the duplex structure,the connecting structure is referred to as a “linker.” The RNA strandsmay have the same or a different number of nucleotides. The maximumnumber of base pairs is the number of nucleotides in the shortest strandof the dsRNA. In addition to the duplex structure, a dsRNA may compriseone or more nucleotide overhangs. A dsRNA as used herein is alsoreferred to as a “small inhibitory RNA” or “siRNA.”

As used herein, a “nucleotide overhang” refers to the unpairednucleotide or nucleotides that protrude from the duplex structure of adsRNA when a 3′-end of one strand of the dsRNA extends beyond the 5′-endof the other strand, or vice versa. “Blunt” or “blunt end” means thatthere are no unpaired nucleotides at that end of the dsRNA, i.e., nonucleotide overhang. A “blunt ended” dsRNA is a dsRNA that isdouble-stranded over its entire length, i.e., no nucleotide overhang ateither end of the molecule.

The term “antisense strand” refers to the strand of a dsRNA whichincludes a region that is substantially complementary to thecorresponding sequence of a target sequence. As used herein, the term“region of complementarity” refers to the region on the antisense strandthat is substantially complementary to a sequence, for example a targetsequence, as defined herein. Where the region of complementarity is notfully complementary to the target sequence, the mismatches may be in theinternal or terminal regions of the molecule. Generally, the mosttolerated mismatches are in the terminal regions, e.g., within 6, 5, 4,3, or 2 nucleotides of the 5′ and/or 3′ terminus.

The term “sense strand,” as used herein, refers to the strand of a dsRNAthat includes a region that is substantially complementary to a regionof the antisense strand.

The term “identity” is the relationship between two or morepolynucleotide sequences, as determined by comparing the sequences.Identity also means the degree of sequence relatedness betweenpolynucleotide sequences, as determined by the match between strings ofsuch sequences. While there exist a number of methods to measureidentity between two polynucleotide sequences, the term is well known toskilled artisans (see, e.g., Sequence Analysis in Molecular Biology, vonHeinje, G., Academic Press (1987); and Sequence Analysis Primer,Gribskov., M. and Devereux, J., eds., M. Stockton Press, New York(1991)). “Substantially identical,” as used herein, means there is avery high degree of homology (preferably 100% sequence identity) betweenthe sense strand of the dsRNA and the corresponding part of the targetgene. However, dsRNA having greater than 90% or 95% sequence identitymay be used in the present invention, and thus sequence variations thatmight be expected due to genetic mutation, strain polymorphism, orevolutionary divergence can be tolerated. Although 100% identity istypical, the dsRNA may contain single or multiple base-pair randommismatches between the RNA and the target gene.

As used herein, the term “SNALP” refers to a stable nucleic acid-lipidparticle. A SNALP represents a vesicle of lipids coating a reducedaqueous interior comprising a nucleic acid such as an iRNA agent or aplasmid from which an iRNA agent is transcribed. SNALPs are described,e.g., in U.S. Patent Application Publication Nos. 20060240093,20070135372, and U.S. Ser. No. 61/045,228 filed Apr. 15, 2008. Theseapplications are hereby incorporated by reference.

“Introducing into a cell,” when referring to a dsRNA, means facilitatinguptake or absorption into the cell, as is understood by those skilled inthe art. Absorption or uptake of dsRNA can occur through unaideddiffusive or active cellular processes, or by auxiliary agents ordevices. The meaning of this term is not limited to cells in vitro; adsRNA may also be “introduced into a cell,” wherein the cell is part ofa living organism. In such instance, introduction into the cell willinclude the delivery to the organism. For example, for in vivo delivery,dsRNA can be injected into a tissue site or administered systemically.In vivo delivery can also be by a beta-glucan delivery system, such asthose described in U.S. Pat. Nos. 5,032,401 and 5,607,677, and U.S.Publication No. 2005/0281781. U.S. Pat. Nos. 5,032,401 and 5,607,677,and U.S. Publication No. 2005/0281781 are hereby incorporated byreference in their entirety. In vitro introduction into a cell includesmethods known in the art such as electroporation and lipofection.

The terms “silence” and “inhibit the expression of,” “down-regulate theexpression of,” “suppress the expression of,” and the like, in as far asthey refer to the CD45 gene, herein refer to the at least partialsuppression of the expression of the CD45 gene, as manifested by areduction of the amount of CD45 mRNA, which may be isolated from a firstcell or group of cells in which the CD45 gene is transcribed, and whichhas or have been treated such that the expression of the CD45 gene isinhibited, as compared to a second cell or group of cells substantiallyidentical to the first cell or group of cells but which has or have notbeen so treated (control cells). The degree of inhibition is usuallyexpressed in terms of

${\frac{\left( {{mRNA}\mspace{14mu} {in}\mspace{14mu} {control}\mspace{14mu} {cells}} \right) - \left( {{mRNA}\mspace{14mu} {in}\mspace{14mu} {treated}\mspace{14mu} {cells}} \right)}{\left( {{mRNA}\mspace{14mu} {in}\mspace{14mu} {control}\mspace{14mu} {cells}} \right)} \cdot 100}\%$

Alternatively, the degree of inhibition may be given in terms of areduction of a parameter that is functionally linked to CD45 geneexpression, e.g. the amount of protein encoded by the CD45 gene which ispresent on the cell surface, or the number of cells displaying a certainphenotype, e.g apoptosis. In principle, CD45 gene silencing may bedetermined in any cell expressing the CD45, either constitutively or bygenomic engineering, and by any appropriate assay. However, when areference is needed in order to determine whether a given siRNA inhibitsthe expression of the CD45 gene by a certain degree and therefore isencompassed by the instant invention, the assays provided in theExamples below shall serve as such reference.

For example, in certain instances, expression of the CD45 gene issuppressed by at least about 20%, 25%, 30%, 35%, 40%, 45%, or 50% byadministration of the double-stranded oligonucleotide featured in theinvention. In one embodiment, the CD45 gene is suppressed by at leastabout 50%, 60%, or 70% by administration of the double-strandedoligonucleotide featured in the invention. In another embodiment, theCD45 gene is suppressed by at least about 75%, 80%, 90% or 95% byadministration of the double-stranded oligonucleotide featured in theinvention.

The terms “treat,” “treatment,” and the like, refer to relief from oralleviation of an infectious disease or an autoimmune disease. In thecontext of the present invention insofar as it relates to any of theother conditions recited herein below (e.g., a CD45-mediated conditionother than an infectious disease or autoimmune disease), the terms“treat,” “treatment,” and the like mean to relieve or alleviate at leastone symptom associated with such condition, or to slow or reverse theprogression of such condition.

As used herein, the term “CD45-mediated condition or disease” andrelated terms and phrases refer to a condition or disorder characterizedby inappropriate, e.g., greater than normal, CD45 activity.Inappropriate CD45 functional activity might arise as the result of CD45expression in cells which normally do not express CD45 or increased CD45expression (leading to, e.g., autoimmune disease). A CD45-mediatedcondition or disease may be completely or partially mediated byinappropriate CD45 functional activity. However, a CD45-mediatedcondition or disease is one in which modulation of CD45 results in someeffect on the underlying condition or disorder (e.g., a CD45 inhibitorresults in some improvement in patient well-being in at least somepatients).

As used herein, the phrases “therapeutically effective amount” and“prophylactically effective amount” refer to an amount that provides atherapeutic benefit in the treatment, prevention, or management of aninfectious disease or an overt symptom of infection, or an autoimmunedisease. The specific amount that is therapeutically effective can bereadily determined by ordinary medical practitioner, and may varydepending on factors known in the art, such as, e.g. the type ofinfection or autoimmune disease, the patient's history and age, thestage of the disease, and the administration of other agents.

As used herein, a “pharmaceutical composition” includes apharmacologically effective amount of a dsRNA and a pharmaceuticallyacceptable carrier. As used herein, “pharmacologically effectiveamount,” “therapeutically effective amount” or simply “effective amount”refers to that amount of a RNA effective to produce the intendedpharmacological, therapeutic or preventive result. For example, if agiven clinical treatment is considered effective when there is at leasta 25% reduction in a measurable parameter associated with a disease ordisorder, a therapeutically effective amount of a drug for the treatmentof that disease or disorder is the amount necessary to effect at least a25% reduction in that parameter.

The term “pharmaceutically acceptable carrier” refers to a carrier foradministration of a therapeutic agent. Such carriers include, but arenot limited to, saline, buffered saline, dextrose, water, glycerol,ethanol, and combinations thereof. The term specifically excludes cellculture medium. For drugs administered orally, pharmaceuticallyacceptable carriers include, but are not limited to pharmaceuticallyacceptable excipients such as inert diluents, disintegrating agents,binding agents, lubricating agents, sweetening agents, flavoring agents,coloring agents and preservatives. Suitable inert diluents includesodium and calcium carbonate, sodium and calcium phosphate, and lactose,while corn starch and alginic acid are suitable disintegrating agents.Binding agents may include starch and gelatin, while the lubricatingagent, if present, will generally be magnesium stearate, stearic acid ortalc. If desired, the tablets may be coated with a material such asglyceryl monostearate or glyceryl distearate, to delay absorption in thegastrointestinal tract.

As used herein, a “transformed cell” is a cell into which a vector hasbeen introduced from which a dsRNA molecule may be expressed.

II. DOUBLE-STRANDED RIBONUCLEIC ACID (DSRNA)

In one embodiment, the invention provides double-stranded ribonucleicacid (dsRNA) molecules for inhibiting the expression of the CD45 gene ina cell or mammal. The dsRNA includes an antisense strand including aregion of complementarity which is complementary to the correspondingregion of an mRNA formed in the expression of the CD45 gene, and whereinthe region of complementarity is less than 30 nucleotides in length,generally 19-24 nucleotides in length. In another embodiment the dsRNA,upon contact with a cell expressing said CD45 gene, inhibits theexpression of said CD45 gene, e.g., in an assay described herein, e.g.,in a P388D1 cell assay (or an assay based on a similar cell) asdescribed herein, by at least 20%, or preferably by at least 40%. ThedsRNA includes two RNA strands that are sufficiently complementary tohybridize to form a duplex structure. The sense strand includes a regionwhich is complementary to the antisense strand, such that the twostrands hybridize and form a duplex structure when combined undersuitable conditions. Generally, the duplex structure is between 15 and30, more generally between 18 and 25, yet more generally between 19 and24, and most generally between 21 and 23 base pairs in length.Similarly, the region of complementarity to the target sequence isbetween 15 and 30, more generally between 18 and 25, yet more generallybetween 19 and 24, and most generally between 19 and 21 nucleotides inlength. In some embodiments, the dsRNA is between 10 and 15 nucleotidesin length, and in other embodiments, the dsRNA is between 25 and 30nucleotides in length. The dsRNA featured in the invention may furtherinclude one or more single-stranded nucleotide overhang(s).

The dsRNA can be synthesized by standard methods known in the art asfurther discussed below, e.g., by use of an automated DNA synthesizer,such as are commercially available from, for example, Biosearch, AppliedBiosystems, Inc. In one embodiment, the CD45 gene is the human CD45gene. In some embodiments, the antisense strand of the dsRNA includes asense sequence from Tables 2, 4 and 5, and the sense strand of the dsRNAincludes a sense sequence from Tables 2, 4 and 5.

In other embodiments, the dsRNA includes at least one nucleotidesequence selected from the groups of sequences provided in Tables 2, 4and 5. In other embodiments, the dsRNA includes at least two sequencesselected from this group, wherein one of the at least two sequences iscomplementary to another of the at least two sequences, and one of theat least two sequences is substantially complementary to a sequence ofan mRNA generated in the expression of the CD45 gene. Generally, thedsRNA includes two oligonucleotides, wherein one oligonucleotide isdescribed as a sense strand in Tables 2, 4, or 5, and the secondoligonucleotide is described as an antisense strand in Tables 2, 4 or 5.

The skilled person is well aware that dsRNAs including a duplexstructure of between 20 and 23, but specifically 21, base pairs havebeen identified as particularly effective in inducing RNA interference(Elbashir et al., EMBO 2001, 20:6877-6888). However, others have foundthat shorter or longer dsRNAs can be effective as well. In theembodiments described above, by virtue of the nature of theoligonucleotide sequences provided in Tables 2, 4, or 5, the dsRNAsfeatured in the invention can include at least one strand of a length ofminimally 21 nt. It can be reasonably expected that shorter dsRNAsincluding one of the sequences of Table 2, 4 or 5, minus only a fewnucleotides on one or both ends may be similarly effective as comparedto the dsRNAs described above. Hence, dsRNAs including a partialsequence of at least 15, 16, 17, 18, 19, 20, or more contiguousnucleotides from one of the sequences of Table 2, 4 or 5, and differingin their ability to inhibit the expression of the CD45 gene in a FACSassay as described herein below by not more than 5, 10, 15, 20, 25, or30% inhibition from a dsRNA including the full sequence, arecontemplated by the invention.

In addition, the dsRNA agents provided in Tables 2, 4 and 5 identifysites in the CD45 mRNA that are susceptible to RNAi based cleavage. Assuch, the invention further includes dsRNAs that target within thesequence targeted by one of the agents featured in the presentinvention. As used herein, a second dsRNA is said to target within thesequence of a first dsRNA if the second dsRNA cleaves the messageanywhere within the mRNA that is complementary to the antisense strandof the first dsRNA. Such a second agent will generally consist of atleast 15 contiguous nucleotides from one of the sequences provided inTables 2, 4 and 5 coupled to additional nucleotide sequences taken fromthe region contiguous to the selected sequence in the CD45 gene.

The dsRNA featured in the invention can contain one or more mismatchesto the target sequence. In one embodiment, the dsRNA targeting CD45contains no more than 3 mismatches. If the antisense strand of the dsRNAcontains mismatches to a target sequence, it is preferable that the areaof mismatch not be located in the center of the region ofcomplementarity. If the antisense strand of the dsRNA containsmismatches to the target sequence, it is preferable that the mismatch berestricted to 5 nucleotides from either end, for example 5, 4, 3, 2, or1 nucleotide from either the 5′ or 3′ end of the region ofcomplementarity. For example, for a 23 nucleotide dsRNA strand which iscomplementary to a region of the CD45 gene, the dsRNA generally does notcontain any mismatch within the central 13 nucleotides. The methodsdescribed within the invention can be used to determine whether a dsRNAcontaining a mismatch to a target sequence is effective in inhibitingthe expression of the CD45 gene. Consideration of the efficacy of dsRNAswith mismatches in inhibiting expression of the CD45 gene is important,especially if the particular region of complementarity in the CD45 geneis known to have polymorphic sequence variation within the population.

In one embodiment, at least one end of the dsRNA has a single-strandednucleotide overhang of 1 to 4, generally 1 or 2 nucleotides. dsRNAshaving at least one nucleotide overhang have unexpectedly superiorinhibitory properties than their blunt-ended counterparts. In oneembodiment the presence of only one nucleotide overhang strengthens theinterference activity of the dsRNA, without affecting its overallstability. dsRNA having only one overhang has proven particularly stableand effective in vivo, as well as in a variety of cells, cell culturemediums, blood, and serum. Generally, the single-stranded overhang islocated at the 3′-terminal end of the antisense strand or,alternatively, at the 3′-terminal end of the sense strand. The dsRNA mayalso have a blunt end, generally located at the 5′-end of the antisensestrand. Such dsRNAs have improved stability and inhibitory activity,thus allowing administration at low dosages, i.e., less than 5 mg/kgbody weight of the recipient per day. In one embodiment, the antisensestrand of the dsRNA has a 1-10 nucleotide overhang at the 3′ end and/orthe 5′ end. In one embodiment, the sense strand of the dsRNA has a 1-10nucleotide overhang at the 3′ end and/or the 5′ end. In anotherembodiment, one or more of the nucleotides in the overhang is replacedwith a nucleoside thiophosphate.

In yet another embodiment, the dsRNA is chemically modified to enhancestability. The nucleic acids featured in the invention may besynthesized and/or modified by methods well established in the art, suchas those described in “Current protocols in nucleic acid chemistry”,Beaucage, S. L. et al. (Edrs.), John Wiley & Sons, Inc., New York, N.Y.,USA, which is hereby incorporated herein by reference. Specific examplesof dsRNA compounds useful in this invention include dsRNAs containingmodified backbones or no natural internucleoside linkages. As defined inthis specification, dsRNAs having modified backbones include those thatretain a phosphorus atom in the backbone and those that do not have aphosphorus atom in the backbone. For the purposes of this specification,and as sometimes referenced in the art, modified dsRNAs that do not havea phosphorus atom in their internucleoside backbone can also beconsidered to be oligonucleosides.

Modified dsRNA backbones include, for example, phosphorothioates, chiralphosphorothioates, phosphorodithioates, phosphotriesters,aminoalkylphosphotriesters, methyl and other alkyl phosphonatesincluding 3′-alkylene phosphonates and chiral phosphonates,phosphinates, phosphoramidates including 3′-amino phosphoramidate andaminoalkylphosphoramidates, thionophosphoramidates,thionoalkylphosphonates, thionoalkylphosphotriesters, andboranophosphates having normal 3′-5′ linkages, 2′-5′ linked analogs ofthese, and those) having inverted polarity wherein the adjacent pairs ofnucleoside units are linked 3′-5′ to 5′-3′ or 2′-5′ to 5′-2′. Varioussalts, mixed salts and free acid forms are also included.

Representative U.S. patents that teach the preparation of the abovephosphorus-containing linkages include, but are not limited to, U.S.Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,195;5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131;5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925;5,519,126; 5,536,821; 5,541,316; 5,550,111; 5,563,253; 5,571,799;5,587,361; and 5,625,050, each of which is herein incorporated byreference

Modified dsRNA backbones that do not include a phosphorus atom thereinhave backbones that are formed by short chain alkyl or cycloalkylinternucleoside linkages, mixed heteroatoms and alkyl or cycloalkylinternucleoside linkages, or ore or more short chain heteroatomic orheterocyclic internucleoside linkages. These include those havingmorpholino linkages (formed in part from the sugar portion of anucleoside); siloxane backbones; sulfide, sulfoxide and sulfonebackbones; formacetyl and thioformacetyl backbones; methylene formacetyland thioformacetyl backbones; alkene containing backbones; sulfamatebackbones; methyleneimino and methylenehydrazino backbones; sulfonateand sulfonamide backbones; amide backbones; and others having mixed N,O, S and CH₂ component parts.

Representative U.S. patents that teach the preparation of the aboveoligonucleosides include, but are not limited to, U.S. Pat. Nos.5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033;5,64,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967;5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,608,046;5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; and,5,677,439, each of which is herein incorporated by reference.

In other dsRNA mimetics, both the sugar and the internucleoside linkage,i.e., the backbone, of the nucleotide units are replaced with novelgroups. The base units are maintained for hybridization with anappropriate nucleic acid target compound. One such oligomeric compound,a dsRNA mimetic that has been shown to have excellent hybridizationproperties, is referred to as a peptide nucleic acid (PNA). In PNAcompounds, the sugar backbone of a dsRNA is replaced with an amidecontaining backbone, in particular an aminoethylglycine backbone. Thenucleobases are retained and are bound directly or indirectly to azanitrogen atoms of the amide portion of the backbone. Representative U.S.patents that teach the preparation of PNA compounds include, but are notlimited to, U.S. Pat. Nos. 5,539,082; 5,714,331; and 5,719,262, each ofwhich is herein incorporated by reference. Further teaching of PNAcompounds can be found in Nielsen et al., Science, 1991, 254, 1497-1500.

Typical embodiments include dsRNAs with phosphorothioate backbones andoligonucleosides with heteroatom backbones, and in particular—CH₂—NH—CH₂—, —CH₂—N(CH₃)—O—CH₂— [known as a methylene (methylimino) orMMI backbone], —CH₂—O—N(CH₃)—CH₂—, —CH₂—N(CH₃)—N(CH₃)—CH₂— and—N(CH₃)—CH₂—CH₂— [wherein the native phosphodiester backbone isrepresented as —O—P—O—CH₂—] of the above-referenced U.S. Pat. No.5,489,677, and the amide backbones of the above-referenced U.S. Pat. No.5,602,240. Other suitable dsRNAs have morpholino backbone structures ofthe above-referenced U.S. Pat. No. 5,034,506.

Modified dsRNAs may also contain one or more substituted sugar moieties.Typical dsRNAs include one of the following at the 2′ position: OH; F;O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S- or N-alkynyl; orO-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl may besubstituted or unsubstituted C₁ to C₁₀ alkyl or C₂ to C₁₀ alkenyl andalkynyl. Other dsRNAs include O[(CH₂)_(n)O]_(m)CH₃, O(CH₂)_(n)OCH₃,O(CH₂)_(n)NH₂, O(CH₂)_(n)CH₃, O(CH₂)_(n)ONH₂, andO(CH₂)_(n)ON[(CH₂)_(n)CH₃)]₂, where n and m are from 1 to about 10.Other typical dsRNAs include one of the following at the 2′ position: C₁to C₁₀ lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkarylor O-aralkyl, SH, SCH₃, OCN, Cl, Br, CN, CF₃, OCF₃, SOCH₃, SO₂CH₃, ONO₂,NO₂, N₃, NH₂, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino,polyalkylamino, substituted silyl, an RNA cleaving group, a reportergroup, an intercalator, a group for improving the pharmacokineticproperties of an dsRNA, or a group for improving the pharmacodynamicproperties of an dsRNA, and other substituents having similarproperties. In one embodiment, the modification includes2′-methoxyethoxy (2′-O—CH₂CH₂OCH₃, also known as 2′-O-(2-methoxyethyl)or 2′-MOE) (Martin et al., Hely. Chim. Acta, 1995, 78, 486-504), i.e.,an alkoxy-alkoxy group. Another suitable modification includes2′-dimethylaminooxyethoxy, i.e., a O(CH₂)₂ON(CH₃)₂ group, also known as2′-DMAOE, as described in examples hereinbelow, and2′-dimethylaminoethoxyethoxy (also known in the art as2′-O-dimethylaminoethoxyethyl or 2′-DMAEOE), i.e.,2′-O—CH₂—O—CH₂—N(CH₂)₂, also described in examples hereinbelow.

Other suitable modifications include 2′-methoxy (2′-OCH₃),2′-aminopropoxy (2′-OCH₂CH₂CH₂NH₂) and 2′-fluoro (2′-F). Similarmodifications may also be made at other positions on the dsRNA,particularly the 3′ position of the sugar on the 3′ terminal nucleotideor in 2′-5′ linked dsRNAs and the 5′ position of 5′ terminal nucleotide.DsRNAs may also have sugar mimetics such as cyclobutyl moieties in placeof the pentofuranosyl sugar. Representative U.S. patents that teach thepreparation of such modified sugar structures include, but are notlimited to, U.S. Pat. Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044;5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811;5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873;5,646,265; 5,658,873; 5,670,633; and 5,700,920, certain of which arecommonly owned with the instant application, and each of which is hereinincorporated by reference in its entirety.

dsRNAs may also include nucleobase (often referred to in the art simplyas “base”) modifications or substitutions. As used herein, “unmodified”or “natural” nucleobases include the purine bases adenine (A) andguanine (G), and the pyrimidine bases thymine (T), cytosine (C) anduracil (U). Modified nucleobases include other synthetic and naturalnucleobases such as 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine,xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkylderivatives of adenine and guanine, 2-propyl and other alkyl derivativesof adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine,5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil,cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo,8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl anal other 8-substitutedadenines and guanines, 5-halo, particularly 5-bromo, 5-trifluoromethyland other 5-substituted uracils and cytosines, 7-methylguanine and7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and7-daazaadenine and 3-deazaguanine and 3-deazaadenine. Furthernucleobases include those disclosed in U.S. Pat. No. 3,687,808, thosedisclosed in The Concise Encyclopedia Of Polymer Science AndEngineering, pages 858-859, Kroschwitz, J. L, ed. John Wiley & Sons,1990, these disclosed by Englisch et al., Angewandte Chemie,International Edition, 1991, 30, 613, and those disclosed by Sanghvi, YS., Chapter 15, DsRNA Research and Applications, pages 289-302, Crooke,S. T. and Lebleu, B., Ed., CRC Press, 1993. Certain of these nucleobasesare particularly useful for increasing the binding affinity of theoligomeric compounds featured in the invention. These include5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6substituted purines, including 2-aminopropyladenine, 5-propynyluraciland 5-propynylcytosine. 5-methylcytosine substitutions have been shownto increase nucleic acid duplex stability by 0.6-1.2° C. (Sanghvi, Y.S., Crooke, S. T. and Lebleu, B., Eds., DsRNA Research and Applications,CRC Press, Boca Raton, 1993, pp. 276-278) and are suitable basesubstitutions, particularly when combined with 2′-O-methoxyethyl sugarmodifications.

Representative U.S. patents that teach the preparation of certain of theabove noted modified nucleobases as well as other modified nucleobasesinclude, but are not limited to, the above noted U.S. Pat. No.3,687,808, as well as U.S. Pat. Nos. 4,845,205; 5,130,30; 5,134,066;5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908;5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121, 5,596,091;5,614,617; and 5,681,941, each of which is herein incorporated byreference, and U.S. Pat. No. 5,750,692, also herein incorporated byreference.

Another modification of the dsRNAs featured in the invention involveschemically linking to the dsRNA one or more moieties or conjugates whichenhance the activity, cellular distribution or cellular uptake of thedsRNA. Such moieties include but are not limited to lipid moieties suchas a cholesterol moiety (Letsinger et al., Proc. Natl. Acid. Sci. USA,199, 86, 6553-6556), cholic acid (Manoharan et al., Biorg. Med. Chem.Let., 1994 4 1053-1060), a thioether, e.g., beryl-5-tritylthiol(Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660, 306-309; Manoharanet al., Biorg. Med. Chem. Let., 1993, 3, 2765-2770), a thiocholesterol(Oberhauser et al., Nucl. Acids Res., 1992, 20, 533-538), an aliphaticchain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al.,EMBO J, 1991, 10, 1111-1118; Kabanov et al., FEBS Lett., 1990, 259,327-330; Svinarchuk et al., Biochimie, 1993, 75, 49-54), a phospholipid,e.g., di-hexadecyl-rac-glycerol or triethyl-ammonium1,2-di-O-hexadecyl-rac-glycero-3-Hphosphonate (Manoharan et al.,Tetrahedron Lett., 1995, 36, 3651-3654; Shea et al., Nucl. Acids Res.,1990, 18, 3777-3783), a polyamine or a polyethylene glycol chain(Manoharan et al., Nucleosides & Nucleotides, 1995, 14, 969-973), oradamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36,3651-3654), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta,1995, 1264, 229-237), or an octadecylamine orhexylamino-carbonyloxycholesterol moiety (Crooke et al., J. Pharmacol.Exp. Ther., 1996, 277, 923-937).

Representative U.S. patents that teach the preparation of such dsRNAconjugates include, but are not limited to, U.S. Pat. Nos. 4,828,979;4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538;5,578,717, 5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045;5,414,077; 5,486,603; 5,512,439; 5,578,718; 5,608,046; 4,587,044;4,605,735; 4,667,025; 4,762,779; 4,789,737; 4,824,941; 4,835,263;4,876,335; 4,904,582; 4,958,013; 5,082,830; 5,112,963; 5,214,136;5,082,830; 5,112,963; 5,214,136; 5,245,022; 5,254,469; 5,258,506;5,262,536; 5,272,250; 5,292,873; 5,317,098; 5,371,241, 5,391,723;5,416,203, 5,451,463; 5,510,475; 5,512,667; 5,514,785; 5,565,552;5,567,810; 5,574,142; 5,585,481; 5,587,371; 5,595,726; 5,597,696;5,599,923; 5,599,928 and 5,688,941, each of which is herein incorporatedby reference.

It is not necessary for all positions in a given compound to beuniformly modified, and in fact more than one of the aforementionedmodifications may be incorporated in a single compound or even at asingle nucleoside within a dsRNA. The present invention also includesdsRNA compounds which are chimeric compounds. “Chimeric” dsRNA compoundsor “chimeras,” in the context of this invention, are dsRNA compounds,particularly dsRNAs, which contain two or more chemically distinctregions, each made up of at least one monomer unit, i.e., a nucleotidein the case of a dsRNA compound. These dsRNAs typically contain at leastone region wherein the dsRNA is modified so as to confer upon the dsRNAincreased resistance to nuclease degradation, increased cellular uptake,and/or increased binding affinity for the target nucleic acid. Anadditional region of the dsRNA may serve as a substrate for enzymescapable of cleaving RNA:DNA or RNA:RNA hybrids. By way of example, RNaseH is a cellular endonuclease which cleaves the RNA strand of an RNA:DNAduplex. Activation of RNase H, therefore, results in cleavage of the RNAtarget, thereby greatly enhancing the efficiency of dsRNA inhibition ofgene expression. Consequently, comparable results can often be obtainedwith shorter dsRNAs when chimeric dsRNAs are used, compared tophosphorothioate deoxydsRNAs hybridizing to the same target region.Cleavage of the RNA target can be routinely detected by gelelectrophoresis and, if necessary, associated nucleic acid hybridizationtechniques known in the art.

In certain instances, the dsRNA may be modified by a non-ligand group. Anumber of non-ligand molecules have been conjugated to dsRNAs in orderto enhance the activity, cellular distribution or cellular uptake of thedsRNA, and procedures for performing such conjugations are available inthe scientific literature. Such non-ligand moieties have included lipidmoieties, such as cholesterol (Letsinger et al., Proc. Natl. Acad. Sci.USA, 1989, 86:6553), cholic acid (Manoharan et al., Bioorg. Med. Chem.Lett., 1994, 4:1053), a thioether, e.g., hexyl-5-tritylthiol (Manoharanet al., Ann. N.Y. Acad. Sci., 1992, 660:306; Manoharan et al., Bioorg.Med. Chem. Let., 1993, 3:2765), a thiocholesterol (Oberhauser et al.,Nucl. Acids Res., 1992, 20:533), an aliphatic chain, e.g., dodecandiolor undecyl residues (Saison-Behmoaras et al., EMBO J., 1991, 10:111;Kabanov et al., FEBS Lett., 1990, 259:327; Svinarchuk et al., Biochimie,1993, 75:49), a phospholipid, e.g., di-hexadecyl-rac-glycerol ortriethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate(Manoharan et al., Tetrahedron Lett., 1995, 36:3651; Shea et al., Nucl.Acids Res., 1990, 18:3777), a polyamine or a polyethylene glycol chain(Manoharan et al., Nucleosides & Nucleotides, 1995, 14:969), oradamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995,36:3651), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta,1995, 1264:229), or an octadecylamine orhexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol.Exp. Ther., 1996, 277:923). Representative United States patents thatteach the preparation of such dsRNA conjugates have been listed above.Typical conjugation protocols involve the synthesis of dsRNAs bearing anaminolinker at one or more positions of the sequence. The amino group isthen reacted with the molecule being conjugated using appropriatecoupling or activating reagents. The conjugation reaction may beperformed either with the dsRNA still bound to the solid support orfollowing cleavage of the dsRNA in solution phase. Purification of thedsRNA conjugate by HPLC typically affords the pure conjugate. The use ofa cholesterol conjugate is particularly suitable since such a moiety canincrease targeting vaginal epithelium cells, a site of CD45 expression.

Vector Encoded dsRNA Agents

The dsRNA featured in the invention can also be expressed fromrecombinant viral vectors intracellularly in vivo. The recombinant viralvectors featured in the invention comprise sequences encoding the dsRNAand any suitable promoter for expressing the dsRNA sequences. Suitablepromoters include, for example, the U6 or H1 RNA pol III promotersequences and the cytomegalovirus promoter. Selection of other suitablepromoters is within the skill in the art. The recombinant viral vectorscan also comprise inducible or regulatable promoters for expression ofthe dsRNA in a particular tissue or in a particular intracellularenvironment. The use of recombinant viral vectors to deliver dsRNA tocells in vivo is discussed in more detail below.

dsRNA featured in the invention can be expressed from a recombinantviral vector either as two separate, complementary RNA molecules, or asa single RNA molecule with two complementary regions.

Any viral vector capable of accepting the coding sequences for the dsRNAmolecule(s) to be expressed can be used, for example vectors derivedfrom adenovirus (AV); adeno-associated virus (AAV); retroviruses (e.g,lentiviruses (LV), Rhabdoviruses, murine leukemia virus); herpes virus,and the like. The tropism of viral vectors can be modified bypseudotyping the vectors with envelope proteins or other surfaceantigens from other viruses, or by substituting different viral capsidproteins, as appropriate.

For example, lentiviral vectors can be pseudotyped with surface proteinsfrom vesicular stomatitis virus (VSV), rabies, Ebola, Mokola, and thelike. AAV vectors can be made to target different cells by engineeringthe vectors to express different capsid protein serotypes. For example,an AAV vector expressing a serotype 2 capsid on a serotype 2 genome iscalled AAV 2/2. This serotype 2 capsid gene in the AAV 2/2 vector can bereplaced by a serotype 5 capsid gene to produce an AAV 2/5 vector.Techniques for constructing AAV vectors which express different capsidprotein serotypes are within the skill in the art; see, e.g., RabinowitzJ E et al. (2002), J Virol 76:791-801, the entire disclosure of which isherein incorporated by reference.

Selection of recombinant viral vectors suitable for use in theinvention, methods for inserting nucleic acid sequences for expressingthe dsRNA into the vector, and methods of delivering the viral vector tothe cells of interest are within the skill in the art. See, for example,Dornburg R (1995), Gene Therap. 2: 301-310; Eglitis M A (1988),Biotechniques 6: 608-614; Miller A D (1990), Hum Gene Therap. 1: 5-14;Anderson W F (1998), Nature 392: 25-30; and Rubinson D A et al., Nat.Genet. 33: 401-406, the entire disclosures of which are hereinincorporated by reference.

Typical viral vectors are those derived from AV and AAV. In oneembodiment, the dsRNA featured in the invention is expressed as twoseparate, complementary single-stranded RNA molecules from a recombinantAAV vector including, for example, either the U6 or H1 RNA promoters, orthe cytomegalovirus (CMV) promoter.

A suitable AV vector for expressing the dsRNA featured in the invention,a method for constructing the recombinant AV vector, and a method fordelivering the vector into target cells, are described in Xia H et al.(2002), Nat. Biotech. 20: 1006-1010.

Suitable AAV vectors for expressing the dsRNA featured in the invention,methods for constructing the recombinant AV vector, and methods fordelivering the vectors into target cells are described in Samulski R etal. (1987), J. Virol. 61: 3096-3101; Fisher K J et al. (1996), J. Virol,70: 520-532; Samulski R et al. (1989), J. Virol. 63: 3822-3826; U.S.Pat. No. 5,252,479; U.S. Pat. No. 5,139,941; International PatentApplication No. WO 94/13788; and International Patent Application No. WO93/24641, the entire disclosures of which are herein incorporated byreference.

III. PHARMACEUTICAL COMPOSITIONS INCLUDING DSRNA

The invention provides pharmaceutical compositions including a dsRNA, asdescribed herein, and a pharmaceutically acceptable carrier. Thepharmaceutical composition including the dsRNA is useful for treating adisease or disorder associated with the expression or activity of theCD45 gene, such as pathological processes mediated by CD45 expression.Such pharmaceutical compositions are formulated based on the mode ofdelivery. One example is compositions that are formulated for systemicadministration via parenteral delivery.

The pharmaceutical compositions featured in the invention areadministered in dosages sufficient to inhibit expression of the CD45gene. The present inventors have found that, because of their improvedefficiency, compositions including the dsRNA can be administered atsurprisingly low dosages. Dosages of 0.6 mg or greater of dsRNA perkilogram body weight of recipient per day is sufficient to suppressexpression of the CD45 gene by greater than 35%, with higher dosagescapable of achieving 65% reduction in expression of the CD45 gene.

In general, a suitable dose of dsRNA will be in the range of 0.01 to200.0 milligrams per kilogram body weight of the recipient per day,generally in the range of 0.02 to 50 mg per kilogram body weight perday. For example, the dsRNA can be administered at 0.01, 0.1, 0.05mg/kg, 0.5 mg/kg, 1 mg/kg, 2 mg/kg, 3 mg/kg, 5 mg/kg, 10 mg/kg, 20mg/kg, 30 mg/kg, 40 mg/kg, or 50 mg/kg per single dose. Thepharmaceutical composition may be administered once daily, or the dsRNAmay be administered as two, three, or more sub-doses at appropriateintervals throughout the day or even using continuous infusion ordelivery through a controlled release formulation. In that case, thedsRNA contained in each sub-dose must be correspondingly smaller inorder to achieve the total daily dosage. The dosage unit can also becompounded for delivery over several days, e.g., using a conventionalsustained release formulation which provides sustained release of thedsRNA over a several day period. Sustained release formulations are wellknown in the art and are particularly useful for vaginal delivery ofagents, such as could be used with the dsRNAs featured in the invention.In this embodiment, the dosage unit contains a corresponding multiple ofthe daily dose.

The skilled artisan will appreciate that certain factors may influencethe dosage and timing required to effectively treat a subject, includingbut not limited to the severity of the disease or disorder, previoustreatments, the general health and/or age of the subject, and otherdiseases present. Moreover, treatment of a subject with atherapeutically effective amount of a composition can include a singletreatment or a series of treatments. Estimates of effective dosages andin vivo half-lives for the individual dsRNAs encompassed by theinvention can be made using conventional methodologies or on the basisof in vivo testing using an appropriate animal model, as describedelsewhere herein.

Advances in mouse genetics have generated a number of mouse models forthe study of various human diseases, such as pathological processesmediated by CD45 expression. Such models are used for in vivo testing ofdsRNA, as well as for determining a therapeutically effective dose.

The present invention also includes pharmaceutical compositions andformulations which include dsRNA targeting CD45. The pharmaceuticalcompositions may be administered in a number of ways depending uponwhether local or systemic treatment is desired and upon the area to betreated. Administration may be topical (e.g., by a transdermal patch),pulmonary (e.g., by inhalation or insufflation of powders or aerosols,including by nebulizer; intratracheal, intranasal, epidermal andtransdermal), oral or parenteral. Administration may also be designed toresult in preferential localization to particular tissues through localdelivery, e.g., by direct intraarticular injection into joints, byrectal administration for direct delivery to the gut and intestines, byintravaginal administration for delivery to the cervix and vagina, byintravitreal administration for delivery to the eye. Parenteraladministration includes intravenous, intraarterial, intraarticular,subcutaneous, intraperitoneal or intramuscular injection or infusion;subdermal, e.g., via an implanted device; or intracranial, e.g., byintrathecal or intraventricular administration.

Pharmaceutical compositions and formulations for topical administrationmay include transdermal patches, ointments, lotions, creams, gels,drops, suppositories, sprays, liquids and powders. Conventionalpharmaceutical carriers, aqueous, powder or oily bases, thickeners andthe like may be necessary or desirable. Coated condoms, gloves and thelike may also be useful. Typical topical formulations include those inwhich the dsRNAs featured in the invention are in admixture with atopical delivery agent such as lipids, liposomes, fatty acids, fattyacid esters, steroids, chelating agents and surfactants. Typical lipidsand liposomes include neutral (e.g., dioleoylphosphatidyl DOPEethanolamine, dimyristoylphosphatidyl choline DMPC,distearolyphosphatidyl choline) negative (e.g., dimyristoylphosphatidylglycerol DMPG), and cationic (e.g., dioleoyltetramethylaminopropyl DOTAPand dioleoylphosphatidyl ethanolamine DOTMA). DsRNAs featured theinvention may be encapsulated within liposomes or may form complexesthereto, in particular to cationic liposomes. Alternatively, dsRNAs maybe complexed to lipids, in particular to cationic lipids. Typical fattyacids and esters include but are not limited arachidonic acid, oleicacid, eicosanoic acid, lauric acid, caprylic acid, capric acid, myristicacid, palmitic acid, stearic acid, linoleic acid, linolenic acid,dicaprate, tricaprate, monoolein, dilaurin, glyceryl 1-monocaprate,1-dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or aC₁₋₁₀ alkyl ester (e.g., isopropylmyristate IPM), monoglyceride,diglyceride or pharmaceutically acceptable salt thereof. Topicalformulations are described in detail in U.S. patent application Ser. No.09/315,298 filed May 20, 1999, which is incorporated herein by referencein its entirety.

In one embodiment, a dsRNA featured in the invention is fullyencapsulated in the lipid formulation (e.g., to form a SPLP, pSPLP,SNALP, or other nucleic acid-lipid particle). As used herein, the term“SNALP” refers to a stable nucleic acid-lipid particle, including SPLP.As used herein, the term “SPLP” refers to a nucleic acid-lipid particlecomprising plasmid DNA encapsulated within a lipid vesicle. SNALPs andSPLPs typically contain a cationic lipid, a non-cationic lipid, and alipid that prevents aggregation of the particle (e.g., a PEG-lipidconjugate). SNALPs and SPLPs are extremely useful for systemicapplications, as they exhibit extended circulation lifetimes followingintravenous (i.v.) injection and accumulate at distal sites (e.g., sitesphysically separated from the administration site). SPLPs include“pSPLP,” which include an encapsulated condensing agent-nucleic acidcomplex as set forth in PCT Publication No. WO 00/03683. The particlesof the present invention typically have a mean diameter of about 50 nmto about 150 nm, more typically about 60 nm to about 130 nm, moretypically about 70 nm to about 110 nm, most typically about 70 to about90 nm, and are substantially nontoxic.

In addition, the nucleic acids when present in the nucleic acid-lipidparticles of the present invention are resistant in aqueous solution todegradation with a nuclease. Nucleic acid-lipid particles and theirmethod of preparation are disclosed in, e.g., U.S. Pat. Nos. 5,976,567;5,981,501; 6,534,484; 6,586,410; 6,815,432; and PCT Publication No. WO96/40964.

In one embodiment, the lipid to drug ratio (mass/mass ratio) (e.g.,lipid to dsRNA ratio) will be in the range of from about 1:1 to about50:1, from about 1:1 to about 25:1, from about 3:1 to about 15:1, fromabout 4:1 to about 10:1, from about 5:1 to about 9:1, or about 6:1 toabout 9:1.

The cationic lipid may be, for example, N,N-dioleyl-N,N-dimethylammoniumchloride (DODAC), N,N-distearyl-N,N-dimethylammonium bromide (DDAB),N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP),N-(1-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA),N,N-dimethyl-2,3-dioleyloxy)propylamine (DODMA),1,2-DiLinoleyloxy-N,N-dimethylaminopropane (DLinDMA),1,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA),1,2-Dilinoleylcarbamoyloxy-3-dimethylaminopropane (DLin-C-DAP),1,2-Dilinoleyoxy-3-(dimethylamino)acetoxypropane (DLin-DAC),1,2-Dilinoleyoxy-3-morpholinopropane (DLin-MA),1,2-Dilinoleoyl-3-dimethylaminopropane (DLinDAP),1,2-Dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA),1-Linoleoyl-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP),1,2-Dilinoleyloxy-3-trimethylaminopropane chloride salt (DLin-TMA.Cl),1,2-Dilinoleoyl-3-trimethylaminopropane chloride salt (DLin-TAP.Cl),1,2-Dilinoleyloxy-3-(N-methylpiperazino)propane (DLin-MPZ), or3-(N,N-Dilinoleylamino)-1,2-propanediol (DLinAP),3-(N,N-Dioleylamino)-1,2-propanedio (DOAP),1,2-Dilinoleyloxo-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DMA),2,2-Dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), or amixture thereof. The cationic lipid may comprise from about 20 mol % toabout 50 mol % or about 40 mol % of the total lipid present in theparticle. The non-cationic lipid may be an anionic lipid or a neutrallipid including, but not limited to, distearoylphosphatidylcholine(DSPC), dioleoylphosphatidylcholine (DOPC),dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol(DOPG), dipalmitoylphosphatidylglycerol (DPPG),dioleoyl-phosphatidylethanolamine (DOPE),palmitoyloleoylphosphatidylcholine (POPC),palmitoyloleoyl-phosphatidylethanolamine (POPE),dioleoyl-phosphatidylethanolamine4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoylphosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE),distearoyl-phosphatidyl-ethanolamine (DSPE), 16-O-monomethyl PE,16-O-dimethyl PE, 18-1-trans PE,1-stearoyl-2-oleoyl-phosphatidyethanolamine (SOPE), cholesterol, or amixture thereof. The non-cationic lipid may be from about 5 mol % toabout 90 mol %, about 10 mol %, or about 58 mol % if cholesterol isincluded, of the total lipid present in the particle.

The conjugated lipid that inhibits aggregation of particles may be, forexample, a polyethyleneglycol (PEG)-lipid including, without limitation,a PEG-diacylglycerol (DAG), a PEG-dialkyloxypropyl (DAA), aPEG-phospholipid, a PEG-ceramide (Cer), or a mixture thereof. ThePEG-DAA conjugate may be, for example, a PEG-dilauryloxypropyl (Ci₂), aPEG-dimyristyloxypropyl (Ci₄), a PEG-dipalmityloxypropyl (Ci₆), or aPEG-distearyloxypropyl (C]₈). The conjugated lipid that preventsaggregation of particles may be from 0 mol % to about 20 mol % or about2 mol % of the total lipid present in the particle.

In some embodiments, the nucleic acid-lipid particle further includescholesterol at, e.g., about 10 mol % to about 60 mol % or about 48 mol %of the total lipid present in the particle.

In one embodiment, the lipidoid ND98.4HCl (MW 1487) (Formula 1),Cholesterol (Sigma-Aldrich), and PEG-Ceramide C16 (Avanti Polar Lipids)can be used to prepare lipid-siRNA nanoparticles (i.e., LNP01particles). Stock solutions of each in ethanol can be prepared asfollows: ND98, 133 mg/mL; Cholesterol, 25 mg/mL, PEG-Ceramide C16, 100mg/mL. The ND98, Cholesterol, and PEG-Ceramide C16 stock solutions canthen be combined in a, e.g., 42:48:10 molar ratio. The combined lipidsolution can be mixed with aqueous siRNA (e.g., in sodium acetate pH 5)such that the final ethanol concentration is about 35-45% and the finalsodium acetate concentration is about 100-300 mM. Lipid-siRNAnanoparticles typically form spontaneously upon mixing. Depending on thedesired particle size distribution, the resultant nanoparticle mixturecan be extruded through a polycarbonate membrane (e.g., 100 nm cut-off)using, for example, a thermobarrel extruder, such as Lipex Extruder(Northern Lipids, Inc). In some cases, the extrusion step can beomitted. Ethanol removal and simultaneous buffer exchange can beaccomplished by, for example, dialysis or tangential flow filtration.Buffer can be exchanged with, for example, phosphate buffered saline(PBS) at about pH 7, e.g., about pH 6.9, about pH 7.0, about pH 7.1,about pH 7.2, about pH 7.3, or about pH 7.4.

LNP01 formulations are described, e.g., in International ApplicationPublication No. WO 2008/042973, which is hereby incorporated byreference.

Formulations prepared by either the standard or extrusion-free methodcan be characterized in similar manners. For example, formulations aretypically characterized by visual inspection. They should be whitishtranslucent solutions free from aggregates or sediment. Particle sizeand particle size distribution of lipid-nanoparticles can be measured bylight scattering using, for example, a Malvern Zetasizer Nano ZS(Malvern, USA). Particles should be about 20-300 nm, such as 40-100 nmin size. The particle size distribution should be unimodal. The totalsiRNA concentration in the formulation, as well as the entrappedfraction, is estimated using a dye exclusion assay. A sample of theformulated siRNA can be incubated with an RNA-binding dye, such asRibogreen (Molecular Probes) in the presence or absence of a formulationdisrupting surfactant, e.g., 0.5% Triton-X100. The total siRNA in theformulation can be determined by the signal from the sample containingthe surfactant, relative to a standard curve. The entrapped fraction isdetermined by subtracting the “free” siRNA content (as measured by thesignal in the absence of surfactant) from the total siRNA content.Percent entrapped siRNA is typically >85%. For SNALP formulation, theparticle size is at least 30 nm, at least 40 nm, at least 50 nm, atleast 60 nm, at least 70 nm, at least 80 nm, at least 90 nm, at least100 nm, at least 110 nm, and at least 120 nm. The suitable range istypically about at least 50 nm to about at least 110 nm, about at least60 nm to about at least 100 nm, or about at least 80 nm to about atleast 90 nm.

Compositions and formulations for oral administration include powders orgranules, microparticulates, nanoparticulates, suspensions or solutionsin water or non-aqueous media, capsules, gel capsules, sachets, tabletsor minitablets. Thickeners, flavoring agents, diluents, emulsifiers,dispersing aids or binders may be desirable. Typical oral formulationsare those in which dsRNAs featured in the invention are administered inconjunction with one or more penetration enhancers surfactants andchelators. Typical surfactants include fatty acids and/or esters orsalts thereof, bile acids and/or salts thereof. Typical bile acids/saltsinclude chenodeoxycholic acid (CDCA) and ursodeoxychenodeoxycholic acid(UDCA), cholic acid, dehydrocholic acid, deoxycholic acid, glucholicacid, glycholic acid, glycodeoxycholic acid, taurocholic acid,taurodeoxycholic acid, sodium tauro-24,25-dihydro-fusidate and sodiumglycodihydrofusidate. Typical fatty acids include arachidonic acid,undecanoic acid, oleic acid, lauric acid, caprylic acid, capric acid,myristic acid, palmitic acid, stearic acid, linoleic acid, linolenicacid, dicaprate, tricaprate, monoolein, dilaurin, glyceryl1-monocaprate, 1-dodecylazacycloheptan-2-one, an acylcarnitine, anacylcholine, or a monoglyceride, a diglyceride or a pharmaceuticallyacceptable salt thereof (e.g. sodium). Suitable combinations ofpenetration enhancers include, for example, fatty acids/salts incombination with bile acids/salts. One typical combination is the sodiumsalt of lauric acid, capric acid and UDCA. Further penetration enhancersinclude polyoxyethylene-9-lauryl ether, polyoxyethylene-20-cetyl ether.DsRNAs featured in the invention may be delivered orally, in granularform including sprayed dried particles, or complexed to form micro ornanoparticles. DsRNA complexing agents include poly-amino acids;polyimines; polyacrylates; polyalkylacrylates, polyoxethanes,polyalkylcyanoacrylates; cationized gelatins, albumins, starches,acrylates, polyethyleneglycols (PEG) and starches;polyalkylcyanoacrylates; DEAE-derivatized polyimines, pollulans,celluloses and starches. Typical complexing agents include chitosan,N-trimethylchitosan, poly-L-lysine, polyhistidine, polyornithine,polyspermines, protamine, polyvinylpyridine,polythiodiethylaminomethylethylene P(TDAE), polyaminostyrene (e.g.p-amino), poly(methylcyanoacrylate), poly(ethylcyanoacrylate),poly(butylcyanoacrylate), poly(isobutylcyanoacrylate),poly(isohexylcynaoacrylate), DEAE-methacrylate, DEAE-hexylacrylate,DEAE-acrylamide, DEAE-albumin and DEAE-dextran, polymethylacrylate,polyhexylacrylate, poly(D,L-lactic acid), poly(DL-lactic-co-glycolicacid (PLGA), alginate, and polyethyleneglycol (PEG). Oral formulationsfor dsRNAs and their preparation are described in detail in U.S.application. Ser. No. 08/886,829 (filed Jul. 1, 1997), Ser. No.09/108,673 (filed Jul. 1, 1998), Ser. No. 09/256,515 (filed Feb. 23,1999), Ser. No. 09/082,624 (filed May 21, 1998) and Ser. No. 09/315,298(filed May 20, 1999), each of which is incorporated herein by referencein their entirety.

Compositions and formulations for parenteral, intrathecal orintraventricular administration may include sterile aqueous solutionswhich may also contain buffers, diluents and other suitable additivessuch as, but not limited to, penetration enhancers, carrier compoundsand other pharmaceutically acceptable carriers or excipients.

Pharmaceutical compositions featured in the invention include, but arenot limited to, solutions, emulsions, and liposome-containingformulations. These compositions may be generated from a variety ofcomponents that include, but are not limited to, preformed liquids,self-emulsifying solids and self-emulsifying semisolids.

The pharmaceutical formulations featured in the invention, which mayconveniently be presented in unit dosage form, may be prepared accordingto conventional techniques well known in the pharmaceutical industry.Such techniques include the step of bringing into association the activeingredients with the pharmaceutical carrier(s) or excipient(s). Ingeneral, the formulations are prepared by uniformly and intimatelybringing into association the active ingredients with liquid carriers orfinely divided solid carriers or both, and then, if necessary, shapingthe product.

The compositions featured in the invention may be formulated into any ofmany possible dosage forms such as, but not limited to, tablets,capsules, gel capsules, liquid syrups, soft gels, suppositories, andenemas. The compositions featured in the invention may also beformulated as suspensions in aqueous, non-aqueous or mixed media.Aqueous suspensions may further contain substances which increase theviscosity of the suspension including, for example, sodiumcarboxymethylcellulose, sorbitol and/or dextran. The suspension may alsocontain stabilizers.

In one embodiment, the pharmaceutical composition may be formulated andused as a foam. Pharmaceutical foams include formulations such as, butnot limited to, emulsions, microemulsions, creams, jellies andliposomes. While basically similar in nature these formulations vary inthe components and the consistency of the final product. The preparationof such compositions and formulations is generally known to thoseskilled in the pharmaceutical and formulation arts and may be applied tothe formulation of the compositions featured herein.

Emulsions

The compositions featured in the invention may be prepared andformulated as emulsions. Emulsions are typically heterogenous systems ofone liquid dispersed in another in the form of droplets usuallyexceeding 0.1 μm in diameter (Idson, in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., NewYork, N.Y., volume 1, p. 199; Rosoff, in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., NewYork, N.Y., Volume 1, p. 245; Block in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., NewYork, N.Y., volume 2, p. 335; Higuchi et al., in Remington'sPharmaceutical Sciences, Mack Publishing Co., Easton, Pa., 1985, p.301). Emulsions are often biphasic systems including two immiscibleliquid phases intimately mixed and dispersed with each other. Ingeneral, emulsions may be of either the water-in-oil (w/o) or theoil-in-water (o/w) variety. When an aqueous phase is finely divided intoand dispersed as minute droplets into a bulk oily phase, the resultingcomposition is called a water-in-oil (w/o) emulsion. Alternatively, whenan oily phase is finely divided into and dispersed as minute dropletsinto a bulk aqueous phase, the resulting composition is called anoil-in-water (o/w) emulsion. Emulsions may contain additional componentsin addition to the dispersed phases, and the active drug which may bepresent as a solution in either the aqueous phase, oily phase or itselfas a separate phase. Pharmaceutical excipients such as emulsifiers,stabilizers, dyes, and anti-oxidants may also be present in emulsions asneeded. Pharmaceutical emulsions may also be multiple emulsions that arecomprised of more than two phases such as, for example, in the case ofoil-in-water-in-oil (o/w/o) and water-in-oil-in-water (w/o/w) emulsions.Such complex formulations often provide certain advantages that simplebinary emulsions do not. Multiple emulsions in which individual oildroplets of an o/w emulsion enclose small water droplets constitute aw/o/w emulsion. Likewise a system of oil droplets enclosed in globulesof water stabilized in an oily continuous phase provides an o/w/oemulsion.

Emulsions are characterized by little or no thermodynamic stability.Often, the dispersed or discontinuous phase of the emulsion is welldispersed into the external or continuous phase and maintained in thisform through the means of emulsifiers or the viscosity of theformulation. Either of the phases of the emulsion may be a semisolid ora solid, as is the case of emulsion-style ointment bases and creams.Other means of stabilizing emulsions entail the use of emulsifiers thatmay be incorporated into either phase of the emulsion. Emulsifiers maybroadly be classified into four categories: synthetic surfactants,naturally occurring emulsifiers, absorption bases, and finely dispersedsolids (Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger andBanker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p.199).

Synthetic surfactants, also known as surface active agents, have foundwide applicability in the formulation of emulsions and have beenreviewed in the literature (Rieger, in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., NewYork, N.Y., volume 1, p. 285; Idson, in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), Marcel Dekker, Inc., New York,N.Y., 1988, volume 1, p. 199). Surfactants are typically amphiphilic andcomprise a hydrophilic and a hydrophobic portion. The ratio of thehydrophilic to the hydrophobic nature of the surfactant has been termedthe hydrophile/lipophile balance (HLB) and is a valuable tool incategorizing and selecting surfactants in the preparation offormulations. Surfactants may be classified into different classes basedon the nature of the hydrophilic group: nonionic, anionic, cationic andamphoteric (Rieger, in Pharmaceutical Dosage Forms, Lieberman, Riegerand Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1,p. 285).

Naturally occurring emulsifiers used in emulsion formulations includelanolin, beeswax, phosphatides, lecithin and acacia. Absorption basespossess hydrophilic properties such that they can soak up water to formw/o emulsions yet retain their semisolid consistencies, such asanhydrous lanolin and hydrophilic petrolatum. Finely divided solids havealso been used as good emulsifiers especially in combination withsurfactants and in viscous preparations. These include polar inorganicsolids, such as heavy metal hydroxides, nonswelling clays such asbentonite, attapulgite, hectorite, kaolin, montmorillonite, colloidalaluminum silicate and colloidal magnesium aluminum silicate, pigmentsand nonpolar solids such as carbon or glyceryl tristearate.

A large variety of non-emulsifying materials are also included inemulsion formulations and contribute to the properties of emulsions.These include fats, oils, waxes, fatty acids, fatty alcohols, fattyesters, humectants, hydrophilic colloids, preservatives and antioxidants(Block, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335;Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199).

Hydrophilic colloids or hydrocolloids include naturally occurring gumsand synthetic polymers such as polysaccharides (for example, acacia,agar, alginic acid, carrageenan, guar gum, karaya gum, and tragacanth),cellulose derivatives (for example, carboxymethylcellulose andcarboxypropylcellulose), and synthetic polymers (for example, carbomers,cellulose ethers, and carboxyvinyl polymers). These disperse or swell inwater to form colloidal solutions that stabilize emulsions by formingstrong interfacial films around the dispersed-phase droplets and byincreasing the viscosity of the external phase.

Since emulsions often contain a number of ingredients such ascarbohydrates, proteins, sterols and phosphatides that may readilysupport the growth of microbes, these formulations often incorporatepreservatives. Commonly used preservatives included in emulsionformulations include methyl paraben, propyl paraben, quaternary ammoniumsalts, benzalkonium chloride, esters of p-hydroxybenzoic acid, and boricacid. Antioxidants are also commonly added to emulsion formulations toprevent deterioration of the formulation. Antioxidants used may be freeradical scavengers such as tocopherols, alkyl gallates, butylatedhydroxyanisole, butylated hydroxytoluene, or reducing agents such asascorbic acid and sodium metabisulfite, and antioxidant synergists suchas citric acid, tartaric acid, and lecithin.

The application of emulsion formulations via dermatological, oral andparenteral routes and methods for their manufacture have been reviewedin the literature (Idson, in Pharmaceutical Dosage Forms, Lieberman,Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y.,volume 1, p. 199). Emulsion formulations for oral delivery have beenvery widely used because of ease of formulation, as well as efficacyfrom an absorption and bioavailability standpoint (Rosoff, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245; Idson, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199). Mineral-oil baselaxatives, oil-soluble vitamins and high fat nutritive preparations areamong the materials that have commonly been administered orally as o/wemulsions.

In one embodiment, the compositions of dsRNAs and nucleic acids areformulated as microemulsions. A microemulsion may be defined as a systemof water, oil and amphiphile which is a single optically isotropic andthermodynamically stable liquid solution (Rosoff, in PharmaceuticalDosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker,Inc., New York, N.Y., volume 1, p. 245). Typically, microemulsions aresystems that are prepared by first dispersing an oil in an aqueoussurfactant solution and then adding a sufficient amount of a fourthcomponent, generally an intermediate chain-length alcohol to form atransparent system. Therefore, microemulsions have also been describedas thermodynamically stable, isotropically clear dispersions of twoimmiscible liquids that are stabilized by interfacial films ofsurface-active molecules (Leung and Shah, in: Controlled Release ofDrugs: Polymers and Aggregate Systems, Rosoff, M., Ed., 1989, VCHPublishers, New York, pages 185-215). Microemulsions commonly areprepared via a combination of three to five components that include oil,water, surfactant, cosurfactant and electrolyte. Whether themicroemulsion is of the water-in-oil (w/o) or an oil-in-water (o/w) typeis dependent on the properties of the oil and surfactant used and on thestructure and geometric packing of the polar heads and hydrocarbon tailsof the surfactant molecules (Schott, in Remington's PharmaceuticalSciences, Mack Publishing Co., Easton, Pa., 1985, p. 271).

The phenomenological approach utilizing phase diagrams has beenextensively studied and has yielded a comprehensive knowledge, to oneskilled in the art, of how to formulate microemulsions (Rosoff, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245; Block, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335). Compared toconventional emulsions, microemulsions offer the advantage ofsolubilizing water-insoluble drugs in a formulation of thermodynamicallystable droplets that are formed spontaneously.

Surfactants used in the preparation of microemulsions include, but arenot limited to, ionic surfactants, non-ionic surfactants, Brij 96,polyoxyethylene oleyl ethers, polyglycerol fatty acid esters,tetraglycerol monolaurate (ML310), tetraglycerol monooleate (M0310),hexaglycerol monooleate (PO310), hexaglycerol pentaoleate (PO500),decaglycerol monocaprate (MCA750), decaglycerol monooleate (MO750),decaglycerol sequioleate (SO750), decaglycerol decaoleate (DAO750),alone or in combination with cosurfactants. The cosurfactant, usually ashort-chain alcohol such as ethanol, 1-propanol, and 1-butanol, servesto increase the interfacial fluidity by penetrating into the surfactantfilm and consequently creating a disordered film because of the voidspace generated among surfactant molecules. Microemulsions may, however,be prepared without the use of cosurfactants and alcohol-freeself-emulsifying microemulsion systems are known in the art. The aqueousphase may typically be, but is not limited to, water, an aqueoussolution of the drug, glycerol, PEG300, PEG400, polyglycerols, propyleneglycols, and derivatives of ethylene glycol. The oil phase may include,but is not limited to, materials such as Captex 300, Captex 355, CapmulMCM, fatty acid esters, medium chain (C8-C12) mono, di, andtri-glycerides, polyoxyethylated glyceryl fatty acid esters, fattyalcohols, polyglycolized glycerides, saturated polyglycolized C8-C10glycerides, vegetable oils and silicone oil.

Microemulsions are particularly of interest from the standpoint of drugsolubilization and the enhanced absorption of drugs. Lipid basedmicroemulsions (both o/w and w/o) have been proposed to enhance the oralbioavailability of drugs, including peptides (Constantinides et al.,Pharmaceutical Research, 1994, 11, 1385-1390; Ritschel, Meth. Find. Exp.Clin. Pharmacol., 1993, 13, 205). Microemulsions afford advantages ofimproved drug solubilization, protection of drug from enzymatichydrolysis, possible enhancement of drug absorption due tosurfactant-induced alterations in membrane fluidity and permeability,ease of preparation, ease of oral administration over solid dosageforms, improved clinical potency, and decreased toxicity (Constantinideset al., Pharmaceutical Research, 1994, 11, 1385; Ho et al., J. Pharm.Sci., 1996, 85, 138-143). Often microemulsions may form spontaneouslywhen their components are brought together at ambient temperature. Thismay be particularly advantageous when formulating thermolabile drugs,peptides or dsRNAs. Microemulsions have also been effective in thetransdermal delivery of active components in both cosmetic andpharmaceutical applications. It is expected that the microemulsioncompositions and formulations featured in the invention will facilitatethe increased systemic absorption of dsRNAs and nucleic acids from thegastrointestinal tract, as well as improve the local cellular uptake ofdsRNAs and nucleic acids within the gastrointestinal tract, vagina,buccal cavity and other areas of administration.

Microemulsions may also contain additional components and additives suchas sorbitan monostearate (Grill 3), Labrasol, and penetration enhancersto improve the properties of the formulation and to enhance theabsorption of the dsRNAs and nucleic acids featured in the invention.Penetration enhancers used in microemulsions may be classified asbelonging to one of five broad categories—surfactants, fatty acids, bilesalts, chelating agents, and non-chelating non-surfactants (Lee et al.,Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92). Eachof these classes has been discussed above.

Liposomes

There are many organized surfactant structures besides microemulsionsthat have been studied and used for the formulation of drugs. Theseinclude monolayers, micelles, bilayers and vesicles. Vesicles, such asliposomes, have attracted great interest because of their specificityand the duration of action they offer from the standpoint of drugdelivery. As used herein, the term “liposome” means a vesicle composedof amphiphilic lipids arranged in a spherical bilayer or bilayers.

Liposomes are unilamellar or multilamellar vesicles which have amembrane formed from a lipophilic material and an aqueous interior. Theaqueous portion contains the composition to be delivered. Cationicliposomes possess the advantage of being able to fuse to the cell wall.Non-cationic liposomes, although not able to fuse as efficiently withthe cell wall, are taken up by macrophages in vivo.

In order to cross intact mammalian skin, lipid vesicles must passthrough a series of fine pores, each with a diameter less than 50 nm,under the influence of a suitable transdermal gradient. Therefore, it isdesirable to use a liposome which is highly deformable and able to passthrough such fine pores.

Further advantages of liposomes include; liposomes obtained from naturalphospholipids are biocompatible and biodegradable; liposomes canincorporate a wide range of water and lipid soluble drugs; liposomes canprotect encapsulated drugs in their internal compartments frommetabolism and degradation (Rosoff, in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., NewYork, N.Y., volume 1, p. 245). Important considerations in thepreparation of liposome formulations are the lipid surface charge,vesicle size and the aqueous volume of the liposomes.

Liposomes are useful for the transfer and delivery of active ingredientsto the site of action. Because the liposomal membrane is structurallysimilar to biological membranes, when liposomes are applied to a tissue,the liposomes start to merge with the cellular membranes and as themerging of the liposome and cell progresses, the liposomal contents areemptied into the cell where the active agent may act.

Liposomal formulations have been the focus of extensive investigation asthe mode of delivery for many drugs. There is growing evidence that fortopical administration, liposomes present several advantages over otherformulations. Such advantages include reduced side-effects related tohigh systemic absorption of the administered drug, increasedaccumulation of the administered drug at the desired target, and theability to administer a wide variety of drugs, both hydrophilic andhydrophobic, into the skin.

Several reports have detailed the ability of liposomes to deliver agentsincluding high-molecular weight DNA into the skin. Compounds includinganalgesics, antibodies, hormones and high-molecular weight DNAs havebeen administered to the skin. The majority of applications resulted inthe targeting of the upper epidermis

Liposomes fall into two broad classes. Cationic liposomes are positivelycharged liposomes which interact with the negatively charged DNAmolecules to form a stable complex. The positively charged DNA/liposomecomplex binds to the negatively charged cell surface and is internalizedin an endosome. Due to the acidic pH within the endosome, the liposomesare ruptured, releasing their contents into the cell cytoplasm (Wang etal., Biochem. Biophys. Res. Commun., 1987, 147, 980-985).

Liposomes which are pH-sensitive or negatively-charged, entrap DNArather than complex with it. Since both the DNA and the lipid aresimilarly charged, repulsion rather than complex formation occurs.Nevertheless, some DNA is entrapped within the aqueous interior of theseliposomes. pH-sensitive liposomes have been used to deliver DNA encodingthe thymidine kinase gene to cell monolayers in culture. Expression ofthe exogenous gene was detected in the target cells (Zhou et al.,Journal of Controlled Release, 1992, 19, 269-274).

One major type of liposomal composition includes phospholipids otherthan naturally-derived phosphatidylcholine. Neutral liposomecompositions, for example, can be formed from dimyristoylphosphatidylcholine (DMPC) or dipalmitoyl phosphatidylcholine (DPPC).Anionic liposome compositions generally are formed from dimyristoylphosphatidylglycerol, while anionic fusogenic liposomes are formedprimarily from dioleoyl phosphatidylethanolamine (DOPE). Another type ofliposomal composition is formed from phosphatidylcholine (PC) such as,for example, soybean PC, and egg PC. Another type is formed frommixtures of phospholipid and/or phosphatidylcholine and/or cholesterol.

Several studies have assessed the topical delivery of liposomal drugformulations to the skin. Application of liposomes containing interferonto guinea pig skin resulted in a reduction of skin herpes sores whiledelivery of interferon via other means (e.g. as a solution or as anemulsion) were ineffective (Weiner et al., Journal of Drug Targeting,1992, 2, 405-410). Further, an additional study tested the efficacy ofinterferon administered as part of a liposomal formulation to theadministration of interferon using an aqueous system, and concluded thatthe liposomal formulation was superior to aqueous administration (duPlessis et al., Antiviral Research, 1992, 18, 259-265).

Non-ionic liposomal systems have also been examined to determine theirutility in the delivery of drugs to the skin, in particular systemsincluding non-ionic surfactant and cholesterol. Non-ionic liposomalformulations including Novasome™ I (glyceryldilaurate/cholesterol/polyoxyethylene-10-stearyl ether) and Novasome™ II(glyceryl distearate/cholesterol/polyoxyethylene-10-stearyl ether) wereused to deliver cyclosporin-A into the dermis of mouse skin. Resultsindicated that such non-ionic liposomal systems were effective infacilitating the deposition of cyclosporin-A into different layers ofthe skin (Hu et al. S. T. P. Pharma. Sci., 1994, 4, 6, 466).

Liposomes also include “sterically stabilized” liposomes, a term which,as used herein, refers to liposomes including one or more specializedlipids that, when incorporated into liposomes, result in enhancedcirculation lifetimes relative to liposomes lacking such specializedlipids. Examples of sterically stabilized liposomes are those in whichpart of the vesicle-forming lipid portion of the liposome (A) includesone or more glycolipids, such as monosialoganglioside G_(M1), or (B) isderivatized with one or more hydrophilic polymers, such as apolyethylene glycol (PEG) moiety. While not wishing to be bound by anyparticular theory, it is thought in the art that, at least forsterically stabilized liposomes containing gangliosides, sphingomyelin,or PEG-derivatized lipids, the enhanced circulation half-life of thesesterically stabilized liposomes derives from a reduced uptake into cellsof the reticuloendothelial system (RES) (Allen et al., FEBS Letters,1987, 223, 42; Wu et al., Cancer Research, 1993, 53, 3765).

Various liposomes including one or more glycolipids are known in theart. Papahadjopoulos et al. (Ann. N.Y. Acad. Sci., 1987, 507, 64)reported the ability of monosialoganglioside G_(M1), galactocerebrosidesulfate and phosphatidylinositol to improve blood half-lives ofliposomes. These findings were expounded upon by Gabizon et al. (Proc.Natl. Acad. Sci. U.S.A., 1988, 85, 6949). U.S. Pat. No. 4,837,028 and WO88/04924, both to Allen et al., disclose liposomes including (1)sphingomyelin and (2) the ganglioside G_(M1) or a galactocerebrosidesulfate ester. U.S. Pat. No. 5,543,152 (Webb et al.) discloses liposomesincluding sphingomyelin. Liposomes including1,2-sn-dimyristoylphosphat-idylcholine are disclosed in WO 97/13499 (Limet al).

Many liposomes including lipids derivatized with one or more hydrophilicpolymers, and methods of preparation thereof, are known in the art.Sunamoto et al. (Bull. Chem. Soc. Jpn., 1980, 53, 2778) describedliposomes including a nonionic detergent, 2C₁₂15G, that contains a PEGmoiety. Illum et al. (FEBS Lett., 1984, 167, 79) noted that hydrophiliccoating of polystyrene particles with polymeric glycols results insignificantly enhanced blood half-lives. Synthetic phospholipidsmodified by the attachment of carboxylic groups of polyalkylene glycols(e.g., PEG) are described by Sears (U.S. Pat. Nos. 4,426,330 and4,534,899). Klibanov et al. (FEBS Lett., 1990, 268, 235) describedexperiments demonstrating that liposomes includingphosphatidylethanolamine (PE) derivatized with PEG or PEG stearate havesignificant increases in blood circulation half-lives. Blume et al.(Biochimica et Biophysica Acta, 1990, 1029, 91) extended suchobservations to other PEG-derivatized phospholipids, e.g., DSPE-PEG,formed from the combination of distearoylphosphatidylethanolamine (DSPE)and PEG. Liposomes having covalently bound PEG moieties on theirexternal surface are described in European Patent No. EP 0 445 131 B1and WO 90/04384 to Fisher. Liposome compositions containing 1-20 molepercent of PE derivatized with PEG, and methods of use thereof, aredescribed by Woodle et al. (U.S. Pat. Nos. 5,013,556 and 5,356,633) andMartin et al. (U.S. Pat. No. 5,213,804 and European Patent No. EP 0 496813 B1). Liposomes including a number of other lipid-polymer conjugatesare disclosed in WO 91/05545 and U.S. Pat. No. 5,225,212 (both to Martinet al.) and in WO 94/20073 (Zalipsky et al.) Liposomes includingPEG-modified ceramide lipids are described in WO 96/10391 (Choi et al).U.S. Pat. No. 5,540,935 (Miyazaki et al.) and U.S. Pat. No. 5,556,948(Tagawa et al.) describe PEG-containing liposomes that can be furtherderivatized with functional moieties on their surfaces.

A limited number of liposomes including nucleic acids are known in theart. WO 96/40062 to Thierry et al. discloses methods for encapsulatinghigh molecular weight nucleic acids in liposomes. U.S. Pat. No.5,264,221 to Tagawa et al. discloses protein-bonded liposomes andasserts that the contents of such liposomes may include an dsRNA RNA.U.S. Pat. No. 5,665,710 to Rahman et al. describes certain methods ofencapsulating oligodeoxynucleotides in liposomes. WO 97/04787 to Love etal. discloses liposomes including dsRNA dsRNAs targeted to the raf gene.

Transfersomes are yet another type of liposomes, and are highlydeformable lipid aggregates which are attractive candidates for drugdelivery vehicles. Transfersomes may be described as lipid dropletswhich are so highly deformable that they are easily able to penetratethrough pores which are smaller than the droplet. Transfersomes areadaptable to the environment in which they are used, e.g. they areself-optimizing (adaptive to the shape of pores in the skin),self-repairing, frequently reach their targets without fragmenting, andoften self-loading. To make transfersomes it is possible to add surfaceedge-activators, usually surfactants, to a standard liposomalcomposition. Transfersomes have been used to deliver serum albumin tothe skin. The transfersome-mediated delivery of serum albumin has beenshown to be as effective as subcutaneous injection of a solutioncontaining serum albumin.

Surfactants find wide application in formulations such as emulsions(including microemulsions) and liposomes. The most common way ofclassifying and ranking the properties of the many different types ofsurfactants, both natural and synthetic, is by the use of thehydrophile/lipophile balance (HLB). The nature of the hydrophilic group(also known as the “head”) provides the most useful means forcategorizing the different surfactants used in formulations (Rieger, inPharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, N.Y., 1988,p. 285).

If the surfactant molecule is not ionized, it is classified as anonionic surfactant. Nonionic surfactants find wide application inpharmaceutical and cosmetic products and are usable over a wide range ofpH values. In general their HLB values range from 2 to about 18depending on their structure. Nonionic surfactants include nonionicesters such as ethylene glycol esters, propylene glycol esters, glycerylesters, polyglyceryl esters, sorbitan esters, sucrose esters, andethoxylated esters. Nonionic alkanolamides and ethers such as fattyalcohol ethoxylates, propoxylated alcohols, and ethoxylated/propoxylatedblock polymers are also included in this class. The polyoxyethylenesurfactants are the most popular members of the nonionic surfactantclass.

If the surfactant molecule carries a negative charge when it isdissolved or dispersed in water, the surfactant is classified asanionic. Anionic surfactants include carboxylates such as soaps, acyllactylates, acyl amides of amino acids, esters of sulfuric acid such asalkyl sulfates and ethoxylated alkyl sulfates, sulfonates such as alkylbenzene sulfonates, acyl isethionates, acyl taurates andsulfosuccinates, and phosphates. The most important members of theanionic surfactant class are the alkyl sulfates and the soaps.

If the surfactant molecule carries a positive charge when it isdissolved or dispersed in water, the surfactant is classified ascationic. Cationic surfactants include quaternary ammonium salts andethoxylated amines. The quaternary ammonium salts are the most usedmembers of this class.

If the surfactant molecule has the ability to carry either a positive ornegative charge, the surfactant is classified as amphoteric. Amphotericsurfactants include acrylic acid derivatives, substituted alkylamides,N-alkylbetaines and phosphatides.

The use of surfactants in drug products, formulations and in emulsionshas been reviewed (Rieger, in Pharmaceutical Dosage Forms, MarcelDekker, Inc., New York, N.Y., 1988, p. 285).

Penetration Enhancers

In one embodiment, various penetration enhancers are used to effect theefficient delivery of nucleic acids, particularly dsRNAs, to the skin ofanimals. Most drugs are present in solution in both ionized andnonionized forms. However, usually only lipid soluble or lipophilicdrugs readily cross cell membranes. It has been discovered that evennon-lipophilic drugs may cross cell membranes if the membrane to becrossed is treated with a penetration enhancer. In addition to aidingthe diffusion of non-lipophilic drugs across cell membranes, penetrationenhancers also enhance the permeability of lipophilic drugs.

Penetration enhancers may be classified as belonging to one of fivebroad categories, i.e., surfactants, fatty acids, bile salts, chelatingagents, and non-chelating non-surfactants (Lee et al., Critical Reviewsin Therapeutic Drug Carrier Systems, 1991, p. 92). Each of the abovementioned classes of penetration enhancers are described below ingreater detail.

Surfactants (or “surface-active agents”) are chemical entities, whichwhen dissolved in an aqueous solution, reduce the surface tension of thesolution or the interfacial tension between the aqueous solution andanother liquid, with the result that absorption of dsRNAs through themucosa is enhanced. In addition to bile salts and fatty acids, thesepenetration enhancers include, for example, sodium lauryl sulfate,polyoxyethylene-9-lauryl ether and polyoxyethylene-20-cetyl ether) (Leeet al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p.92); and perfluorochemical emulsions, such as FC-43. Takahashi et al.,J. Pharm. Pharmacol., 1988, 40, 252).

Fatty acids: Various fatty acids and their derivatives which act aspenetration enhancers include, for example, oleic acid, lauric acid,capric acid (n-decanoic acid), myristic acid, palmitic acid, stearicacid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein(1-monooleoyl-rac-glycerol), dilaurin, caprylic acid, arachidonic acid,glycerol 1-monocaprate, 1-dodecylazacycloheptan-2-one, acylcarnitines,acylcholines, C₁₋₁₀ alkyl esters thereof (e.g., methyl, isopropyl andt-butyl), and mono- and di-glycerides thereof (i.e., oleate, laurate,caprate, myristate, palmitate, stearate, linoleate, etc.) (Lee et al.,Critical Reviews in Therapeutic Drug Carryier Systems, 1991, p. 92;Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990,7, 1-33; El Hariri et al., J. Pharm. Pharmacol., 1992, 44, 651-654).

Bile salts: The physiological role of bile includes the facilitation ofdispersion and absorption of lipids and fat-soluble vitamins (Brunton,Chapter 38 in: Goodman & Gilman's The Pharmacological Basis ofTherapeutics, 9th Ed., Hardman et al. Eds., McGraw-Hill, New York, 1996,pp. 934-935). Various natural bile salts, and their syntheticderivatives, act as penetration enhancers. Thus the term “bile salts”includes any of the naturally occurring components of bile as well asany of their synthetic derivatives. The bile salts include, for example,cholic acid (or its pharmaceutically acceptable sodium salt, sodiumcholate), dehydrocholic acid (sodium dehydrocholate), deoxycholic acid(sodium deoxycholate), glucholic acid (sodium glucholate), glycholicacid (sodium glycocholate), glycodeoxycholic acid (sodiumglycodeoxycholate), taurocholic acid (sodium taurocholate),taurodeoxycholic acid (sodium taurodeoxycholate), chenodeoxycholic acid(sodium chenodeoxycholate), ursodeoxycholic acid (UDCA), sodiumtauro-24,25-dihydro-fusidate (STDHF), sodium glycodihydrofusidate andpolyoxyethylene-9-lauryl ether (POE) (Lee et al., Critical Reviews inTherapeutic Drug Carrier Systems, 1991, page 92; Swinyard, Chapter 39In: Remington's Pharmaceutical Sciences, 18th Ed., Gennaro, ed., MackPublishing Co., Easton, Pa., 1990, pages 782-783; Muranishi, CriticalReviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33; Yamamoto etal., J. Pharm. Exp. Ther., 1992, 263, 25; Yamashita et al., J. Pharm.Sci., 1990, 79, 579-583).

“Chelating Agents” are defined as compounds that remove metallic ionsfrom solution by forming complexes therewith, with the result thatabsorption of dsRNAs through the mucosa is enhanced. With regards totheir use as penetration enhancers, chelating agents have the addedadvantage of also serving as DNase inhibitors, as most characterized DNAnucleases require a divalent metal ion for catalysis and are thusinhibited by chelating agents (Jarrett, J. Chromatogr., 1993, 618,315-339). Chelating agents include but are not limited to disodiumethylenediaminetetraacetate (EDTA), citric acid, salicylates (e.g.,sodium salicylate, 5-methoxysalicylate and homovanilate), N-acylderivatives of collagen, laureth-9 and N-amino acyl derivatives ofbeta-diketones (enamines) (Lee et al., Critical Reviews in TherapeuticDrug Carrier Systems, 1991, page 92; Muranishi, Critical Reviews inTherapeutic Drug Carrier Systems, 1990, 7, 1-33; Buur et al., J. ControlRel., 1990, 14, 43-51).

Non-chelating non-surfactants: As used herein, non-chelatingnon-surfactant penetration enhancing compounds can be defined ascompounds that demonstrate insignificant activity as chelating agents oras surfactants but that nonetheless enhance absorption of dsRNAs throughthe alimentary mucosa (Muranishi, Critical Reviews in Therapeutic DrugCarrier Systems, 1990, 7, 1-33). This class of penetration enhancersinclude, for example, unsaturated cyclic ureas, 1-alkyl- and1-alkenylazacyclo-alkanone derivatives (Lee et al., Critical Reviews inTherapeutic Drug Carrier Systems, 1991, page 92); and non-steroidalanti-inflammatory agents such as diclofenac sodium, indomethacin andphenylbutazone (Yamashita et al., J. Pharm. Pharmacol., 1987, 39,621-626).

Agents that enhance uptake of dsRNAs at the cellular level may also beadded to the pharmaceutical and other compositions featured in theinvention. For example, cationic lipids, such as lipofectin (Junichi etal, U.S. Pat. No. 5,705,188), cationic glycerol derivatives, andpolycationic molecules, such as polylysine (Lollo et al., PCTApplication WO 97/30731), are also known to enhance the cellular uptakeof dsRNAs.

Other agents may be utilized to enhance the penetration of theadministered nucleic acids, including glycols such as ethylene glycoland propylene glycol, pyrrols such as 2-pyrrol, azones, and terpenessuch as limonene and menthone.

Carriers

Certain compositions featured herein also incorporate carrier compoundsin the formulation. As used herein, “carrier compound” or “carrier” canrefer to a nucleic acid, or analog thereof, which is inert (i.e., doesnot possess biological activity per se), but is recognized as a nucleicacid by in vivo processes that reduce the bioavailability of a nucleicacid having biological activity by, for example, degrading thebiologically active nucleic acid or promoting its removal fromcirculation. The coadministration of a nucleic acid and a carriercompound, typically with an excess of the latter substance, can resultin a substantial reduction of the amount of nucleic acid recovered inthe liver, kidney or other extracirculatory reservoirs, presumably dueto competition between the carrier compound and the nucleic acid for acommon receptor. For example, the recovery of a partiallyphosphorothioate dsRNA in hepatic tissue can be reduced when it iscoadministered with polyinosinic acid, dextran sulfate, polycytidic acidor 4-acetamido-4′isothiocyano-stilbene-2,2′-disulfonic acid (Miyao etal., DsRNA Res. Dev., 1995, 5, 115-121; Takakura et al., DsRNA & Nucl.Acid Drug Dev., 1996, 6, 177-183.

Excipients

In contrast to a carrier compound, a “pharmaceutical carrier” or“excipient” is a pharmaceutically acceptable solvent, suspending agentor any other pharmacologically inert vehicle for delivering one or morenucleic acids to an animal. The excipient may be liquid or solid and isselected, with the planned manner of administration in mind, so as toprovide for the desired bulk, consistency, etc., when combined with anucleic acid and the other components of a given pharmaceuticalcomposition. Typical pharmaceutical carriers include, but are notlimited to, binding agents (e.g., pregelatinized maize starch,polyvinylpyrrolidone or hydroxypropyl methylcellulose, etc.); fillers(e.g., lactose and other sugars, microcrystalline cellulose, pectin,gelatin, calcium sulfate, ethyl cellulose, polyacrylates or calciumhydrogen phosphate, etc.); lubricants (e.g., magnesium stearate, talc,silica, colloidal silicon dioxide, stearic acid, metallic stearates,hydrogenated vegetable oils, corn starch, polyethylene glycols, sodiumbenzoate, sodium acetate, etc.); disintegrants (e.g., starch, sodiumstarch glycolate, etc.); and wetting agents (e.g., sodium laurylsulphate, etc).

Pharmaceutically acceptable organic or inorganic excipients suitable fornon-parenteral administration, and that do not deleteriously react withnucleic acids, can also be used to formulate the compositions featuredin the present invention. Suitable pharmaceutically acceptable carriersinclude, but are not limited to, water, salt solutions, alcohols,polyethylene glycols, gelatin, lactose, amylose, magnesium stearate,talc, silicic acid, viscous paraffin, hydroxymethylcellulose,polyvinylpyrrolidone and the like.

Formulations for topical administration of nucleic acids may includesterile and non-sterile aqueous solutions, non-aqueous solutions incommon solvents such as alcohols, or solutions of the nucleic acids inliquid or solid oil bases. The solutions may also contain buffers,diluents and other suitable additives. Pharmaceutically acceptableorganic or inorganic excipients suitable for non-parenteraladministration which do not deleteriously react with nucleic acids canbe used.

Suitable pharmaceutically acceptable excipients include, but are notlimited to, water, salt solutions, alcohol, polyethylene glycols,gelatin, lactose, amylose, magnesium stearate, talc, silicic acid,viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone and thelike.

Other Components

The compositions featured in the invention may additionally containother adjunct components conventionally found in pharmaceuticalcompositions, at their art-established usage levels. Thus, for example,the compositions may contain additional, compatible,pharmaceutically-active materials such as, for example, antipruritics,astringents, local anesthetics or anti-inflammatory agents, or maycontain additional materials useful in physically formulating variousdosage forms of the compositions featured in the present invention, suchas dyes, flavoring agents, preservatives, antioxidants, opacifiers,thickening agents and stabilizers. However, such materials, when added,should not unduly interfere with the biological activities of thecomponents of the compositions featured in the invention. Theformulations can be sterilized and, if desired, mixed with auxiliaryagents, e.g., lubricants, preservatives, stabilizers, wetting agents,emulsifiers, salts for influencing osmotic pressure, buffers, colorings,flavorings and/or aromatic substances and the like which do notdeleteriously interact with the nucleic acid(s) of the formulation.

Aqueous suspensions may contain substances which increase the viscosityof the suspension including, for example, sodium carboxymethylcellulose,sorbitol and/or dextran. The suspension may also contain stabilizers.

Certain embodiments featured in the invention provide pharmaceuticalcompositions containing (a) one or more antisense compounds and (b) oneor more other therapeutic agents which function by a non-antisensemechanism. For example, the one or more other therapeutic agents includeantibiotic or antiviral agents. Exemplary antibiotics include, e.g.,amphotericin B, norfloxacin, miconazole nitrate, ofloxacin, idoxuridine,chloramphenicol, colistin sodium methanesulfonate, carbenicillin sodium,beta-lactam antibiotics, cefoxitin, n-formanidolthienamycin and otherthienamycin derivatives, tetracyclines, neomycin, carbenicillin,colistin, penicillin G, polymyxin B, vancomycin, cefazolin,cephaloridine, chibrorifamycin, gramicidin, bacitracin and sulfonamides.Exemplary antiviral agents include, e.g., acyclovir and interferon. Forthe treatment of autoimmune disease the one or more other therapeuticagents can include, e.g., interferon beta (e.g., IFNbeta-1a and IFN-1b,gliatriamer acetate (Copaxone), cyclophosphamide, methotrexate,azathioprine (Imuran), cladribine (Leustatin), cyclosporine,mitoxantrone, and glucocorticoids (e.g., adrenocorticotropic hormone(ACTH), methylprednisolone, and dexamethasone). For treatment of Graves'disease, for example, the additional therapeutic agent can be, e.g., anantithyroid drug, such as propylthiouracil (PTU) or methimazole. Theinvention also includes methods of treating a disorder described hereinby administration of a dsRNA described herein and one or more othertherapeutic agents which function by a non-antisense mechanism, e.g.,one or more of the agents listed above. The agents can be administeredin a single formulation or may be administered separately, at the sameor at different times.

Toxicity and therapeutic efficacy of such compounds can be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., for determining the LD50 (the dose lethal to 50% of thepopulation) and the ED50 (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects isthe therapeutic index and it can be expressed as the ratio LD50/ED50.Suitable compounds typically exhibit high therapeutic indices.

The data obtained from cell culture assays and animal studies can beused in formulation a range of dosage for use in humans. The dosage ofcompositions featured in the invention lies generally within a range ofcirculating concentrations that include the ED50 with little or notoxicity. The dosage may vary within this range depending upon thedosage form employed and the route of administration utilized. For anycompound used in the methods featured in the invention, thetherapeutically effective dose can be estimated initially from cellculture assays. A dose may be formulated in animal models to achieve acirculating plasma concentration range of the compound or, whenappropriate, of the polypeptide product of a target sequence (e.g.,achieving a decreased concentration of the polypeptide) that includesthe IC50 (i.e., the concentration of the test compound which achieves ahalf-maximal inhibition of symptoms) as determined in cell culture. Suchinformation can be used to more accurately determine useful doses inhumans. Levels in plasma may be measured, for example, by highperformance liquid chromatography.

In addition to their administration individually or as a plurality, asdiscussed above, the dsRNAs featured in the invention can beadministered in combination with other known agents effective intreatment of pathological processes mediated by CD45 expression. In anyevent, the administering physician can adjust the amount and timing ofdsRNA administration on the basis of results observed using standardmeasures of efficacy known in the art or described herein.

Methods for Treating Diseases Caused by Expression of the CD45 Gene

In one embodiment, the invention provides a method for treating asubject having a pathological condition mediated by the expression ofthe CD45 gene, such as an autoimmune disease or an infectious disease.In this embodiment, the dsRNA acts as a therapeutic agent forcontrolling the expression of the CD45 protein. The method includesadministering a pharmaceutical composition featured in the invention tothe patient (e.g., human), such that expression of the CD45 gene issilenced. Because of their high specificity, the dsRNAs featured in theinvention specifically target mRNAs of the CD45 gene.

As used herein, the term “CD45-mediated condition or disease” andrelated terms and phrases refer to a condition or disorder characterizedby unwanted or inappropriate, e.g., abnormal CD45 activity.Inappropriate CD45 functional activity might arise as the result of CD45expression in cells which normally do not express CD45, increased CD45expression and/or activity (leading to, e.g., autoimmune disorders anddiseases, or increased susceptibility to disease). A CD45-mediatedcondition or disease may be completely or partially mediated byinappropriate CD45 functional activity which may result by way ofinappropriate activation of CD45. Regardless, a CD45-mediated conditionor disease is one in which modulation of CD45 via RNA interferenceresults in some effect on the underlying condition or disorder (e.g., aCD45 inhibitor results in some improvement in patient well-being in atleast some patients).

The anti-CD45 dsRNAs featured in the present invention may be used totreat or diagnose an infection or immune disorder in a subject. Themethods include administering to a subject an anti-CD45 dsRNA in anamount effective to treat an infectious disease or autoimmune disorder.

Pathological processes refer to a category of biological processes thatproduce a deleterious effect. For example, unregulated expression ofCD45 is associated with autoimmunity and infectious disease. A compoundfeatured in the invention can typically modulate a pathological processwhen the compound reduces the degree or severity of the process. Forinstance, autoimmunity or an infection may be prevented or relatedpathological processes can be modulated by the administration ofcompounds that reduce or modulate in some way the expression or at leastone activity CD45.

The dsRNA molecules featured herein may, therefore, be used to treat anautoimmune or infectious disease. Autoimmune diseases that can betreated with a dsRNA that targets CD45 include, but are not limited to,Graves' disease, Hashimoto's thyroiditis, multiple sclerosis, andsystemic sclerosis. In one embodiment, a dsRNA targeting CD45 isadministered to a patient who has received an organ transplant, such asto reduce the risk of damage to the organ (e.g., from ischemia andreperfusion, such as caused by an inflammatory response) and the risk oforgan rejection. Infectious diseases that can be treated with a dsRNAthat targets CD45 include but are not limited to influenza, anthrax,Ebola, human immunodeficiency virus (HIV), vesicular stomatitis virus(VSV), rabies, Mokola, Rous sarcoma virus, and hepatitis, such ashepatitis A, B and C strains.

The pharmaceutical compositions encompassed by the invention may beadministered by any means known in the art including, but not limited tooral or parenteral routes, including intravenous, intramuscular,intraarticular, intraperitoneal, subcutaneous, intravitreal,transdermal, airway (aerosol), nasal, rectal, vaginal and topical(including buccal and sublingual) administration, and epiduraladministration. In some embodiments, the pharmaceutical compositions areadministered intraveneously by infusion or injection.

Before administration of a full dose of a dsRNA targeting CD45, apatient can be administered a smaller dose, such as a 5% infusionreaction, and monitored for adverse effects, such as an allergicreaction. In another example, the patient can be monitored for unwantedimmunostimulatory effects, such as increased cytokine (e.g., TNF-alphaor INF-alpha) levels.

Many CD45-associated diseases and disorders are hereditary. Therefore, apatient in need of a dsRNA targeting CD45 can be identified by taking afamily history. A healthcare provider, such as a doctor, nurse, orfamily member, can take a family history before prescribing oradministering a dsRNA. A DNA test may also be performed on the patientto identify a mutation in the target gene, before a dsRNA isadministered to the patient.

Methods for Inhibiting Expression of the CD45 Gene

In yet another aspect, the invention provides a method for inhibitingthe expression of the CD45 gene in a mammal. The method includesadministering a CD45 dsRNA to the mammal such that expression of thetarget CD45 gene is silenced. Because of their high specificity, thedsRNAs specifically target RNAs (primary or processed) of the targetCD45 gene. Compositions and methods for inhibiting the expression of theCD45 gene using dsRNAs can be performed as described elsewhere herein.

In one embodiment, the method includes administering a compositionincluding a dsRNA, wherein the dsRNA includes a nucleotide sequencewhich is complementary to at least a part of an RNA transcript of theCD45 gene of the mammal to be treated. When the organism to be treatedis a mammal such as a human, the composition may be administered by anymeans known in the art including, but not limited to oral or parenteralroutes, including intravenous, intramuscular, intraarticular,intracranial, subcutaneous, intravitreal, transdermal, airway (aerosol),nasal, rectal, vaginal and topical (including buccal and sublingual)administration. In some embodiments, the compositions are administeredby intraveneous infusion or injection.

TABLE 1 Target positions of duplex dsRNAs Human Reference NM_002838.2sequence of total 23-mer Duplex Name ID # pos. in 23 mer SEQ ID NO:target site AD-14008   69 2206-2228  97 CAGAAUAAAAACCGUUAUGUUGA AD-14009 225 2213-2235  98 AAAACCGUUAUGUUGACAUUCUU AD-14010  211 1927-1949  99UUUCUGAUUAUUGUGACAUCAAU AD-14011  224 2210-2232 100AUAAAAACCGUUAUGUUGACAUU AD-14012  233 2326-2348 101GAACCCAGGAAAUACAUUGCUGC AD-14013  222 2204-2226 102ACCAGAAUAAAAACCGUUAUGUU AD-14014  223 2205-2227 103CCAGAAUAAAAACCGUUAUGUUG AD-14015  235 2409-2431 104CACAGUUAUUGUCAUGGUCACUC AD-14016  454 2207-2229 105AGAAUAAAAACCGUUAUGUUGAC AD-14017  463 2329-2351 106CCCAGGAAAUACAUUGCUGCACA AD-14018 1389 2212-2234 107AAAAACCGUUAUGUUGACAUUCU AD-14019  610 2211-2233 108UAAAAACCGUUAUGUUGACAUUC AD-14020 1094 2215-2237 109AACCGUUAUGUUGACAUUCUUCC AD-14021  611 2214-2236 110AAACCGUUAUGUUGACAUUCUUC AD-14022 1388 2209-2231 111AAUAAAAACCGUUAUGUUGACAU AD-14023 1095 2216-2238 112ACCGUUAUGUUGACAUUCUUCCU AD-14024 1147 2408-2430 113CCACAGUUAUUGUCAUGGUCACU AD-14025  725 1929-1951 114UCUGAUUAUUGUGACAUCAAUAG AD-14026 1364 1930-1952 115CUGAUUAUUGUGACAUCAAUAGC AD-14027 1124 2330-2352 116CCAGGAAAUACAUUGCUGCACAA AD-14028 1050 2028-2050 117UGUUGAAAGGGAUGAUGAAAAAC AD-14029  609 2208-2230 118GAAUAAAAACCGUUAUGUUGACA AD-14030  724 1928-1950 119UUCUGAUUAUUGUGACAUCAAUA AD-14031 1123 2327-2349 120AACCCAGGAAAUACAUUGCUGCA AD-14032 1125 2331-2353 121CAGGAAAUACAUUGCUGCACAAG AD-14033 1146 2407-2429 122GCCACAGUUAUUGUCAUGGUCAC AD-14034 1403 2436-2458 123UGAAGAAGGAAACAGGAACAAGU AD-14035 1365 1931-1953 124UGAUUAUUGUGACAUCAAUAGCC AD-14036 1394 2328-2350 125ACCCAGGAAAUACAUUGCUGCAC AD-14037 1154 2431-2453 126CGAUGUGAAGAAGGAAACAGGAA AD-14038 1022 1932-1954 127GAUUAUUGUGACAUCAAUAGCCC AD-14039 1121 2324-2346 128AAGAACCCAGGAAAUACAUUGCU AD-14040 1122 2325-2347 129AGAACCCAGGAAAUACAUUGCUG AD-14041  760 2405-2427 130AAGCCACAGUUAUUGUCAUGGUC AD-14042 1401 2429-2451 131CUCGAUGUGAAGAAGGAAACAGG AD-14043 1153 2430-2452 132UCGAUGUGAAGAAGGAAACAGGA AD-14044 1402 2432-2454 133GAUGUGAAGAAGGAAACAGGAAC AD-14045 1155 2433-2455 134AUGUGAAGAAGGAAACAGGAACA AD-14046  744 2217-2239 135CCGUUAUGUUGACAUUCUUCCUU AD-14047 1701 2406-2428 136AGCCACAGUUAUUGUCAUGGUCA AD-14048 1704 2439-2461 137AGAAGGAAACAGGAACAAGUGUG AD-14049 1833 2435-2457 138GUGAAGAAGGAAACAGGAACAAG AD-14050 1564 2133-2155 139UCUGGCUGAAUUUCAGAGCAUCC AD-14051 1657 2027-2049 140UUGUUGAAAGGGAUGAUGAAAAA AD-14052 1673 2132-2154 141UUCUGGCUGAAUUUCAGAGCAUC AD-14053 1648 1926-1948 142AUUUCUGAUUAUUGUGACAUCAA AD-14054 1703 2437-2459 143GAAGAAGGAAACAGGAACAAGUG AD-14055 1647 1925-1947 144CAUUUCUGAUUAUUGUGACAUCA

TABLE 2 Sense and Antisense sequences of duplex dsRNAs Sense AntisenseSEQ strand sequence SEQ strand sequence Duplex ID (5′ to 3′) Double ID(5′ to 3′) Double Name NO: Name overhang design NO: Name overhang designAD-14008  1 A22737 GAAuAAAAAccGuuAuGuuTsT  2 A22738AAcAuAACGGUUUUuAUUCTsT AD-14009  3 A22739 AAccGuuAuGuuGAcAuucTsT  4A22740 GAAUGUcAAcAuAACGGUUTsT AD-14010  5 A22741 ucuGAuuAuuGuGAcAucATsT 6 A22742 UGAUGUcAcAAuAAUcAGATsT AD-14011  7 A22743AAAAAccGuuAuGuuGAcATsT  8 A22744 UGUcAAcAuAACGGUUUUUTsT AD-14012  9A22745 AcccAGGAAAuAcAuuGcuTsT 10 A22746 AGcAAUGuAUUUCCUGGGUTsT AD-1401311 A22747 cAGAAuAAAAAccGuuAuGTsT 12 A22748 cAuAACGGUUUUuAUUCUGTsTAD-14014 13 A22749 AGAAuAAAAAccGuuAuGuTsT 14 A22750AcAuAACGGUUUUuAUUCUTsT AD-14015 15 A22751 cAGuuAuuGucAuGGucAcTsT 16A22752 GUGACcAUGAcAAuAACUGTsT AD-14016 17 A22753 AAuAAAAAccGuuAuGuuGTsT18 A22754 cAAcAuAACGGUUUUuAUUTsT AD-14017 19 A22755cAGGAAAuAcAuuGcuGcATsT 20 A22756 UGcAGcAAUGuAUUUCCUGTsT AD-14018 21A22757 AAAccGuuAuGuuGAcAuuTsT 22 A22758 AAUGUcAAcAuAACGGUUUTsT AD-1401923 A22759 AAAAccGuuAuGuuGAcAuTsT 24 A22760 AUGUcAAcAuAACGGUUUUTsTAD-14020 25 A22761 ccGuuAuGuuGAcAuucuuTsT 26 A22762AAGAAUGUcAAcAuAACGGTsT AD-14021 27 A22763 AccGuuAuGuuGAcAuucuTsT 28A22764 AGAAUGUcAAcAuAACGGUTsT AD-14022 29 A22765 uAAAAAccGuuAuGuuGAcTsT30 A22766 GUcAAcAuAACGGUUUUuATsT AD-14023 31 A22767cGuuAuGuuGAcAuucuucTsT 32 A22768 GAAGAAUGUcAAcAuAACGTsT AD-14024 33A22769 AcAGuuAuuGucAuGGucATsT 34 A22770 UGACcAUGAcAAuAACUGUTsT AD-1402535 A22771 uGAuuAuuGuGAcAucAAuTsT 36 A22772 AUUGAUGUcAcAAuAAUcATsTAD-14026 37 A22773 GAuuAuuGuGAcAucAAuATsT 38 A22774uAUUGAUGUcAcAAuAAUCTsT AD-14027 39 A22775 AGGAAAuAcAuuGcuGcAcTsT 40A22776 GUGcAGcAAUGuAUUUCCUTsT AD-14028 41 A22777 uuGAAAGGGAuGAuGAAAATsT42 A22778 UUUUcAUcAUCCCUUUcAATsT AD-14029 43 A22779AuAAAAAccGuuAuGuuGATsT 44 A22780 UcAAcAuAACGGUUUUuAUTsT AD-14030 45A22781 cuGAuuAuuGuGAcAucAATsT 46 A22782 UUGAUGUcAcAAuAAUcAGTsT AD-1403147 A22783 cccAGGAAAuAcAuuGcuGTsT 48 A22784 cAGcAAUGuAUUUCCUGGGTsTAD-14032 49 A22785 GGAAAuAcAuuGcuGcAcATsT 50 A22786UGUGcAGcAAUGuAUUUCCTsT AD-14033 51 A22787 cAcAGuuAuuGucAuGGucTsT 52A22788 GACcAUGAcAAuAACUGUGTsT AD-14034 53 A22789 AAGAAGGAAAcAGGAAcAATsT54 A22790 UuGUUCCuGUUUCCUUCUUTsT AD-14035 55 A22791AuuAuuGuGAcAucAAuAGTsT 56 A22792 CuAUUGAUGUcAcAAuAAUTsT AD-14036 57A22793 ccAGGAAAuAcAuuGcuGcTsT 58 A22794 GcAGcAAUGuAUUUCCUGGTsT AD-1403759 A22795 AuGuGAAGAAGGAAAcAGGTsT 60 A22796 CCUGUUUCCUUCUUcAcAUTsTAD-14038 61 A22797 uuAuuGuGAcAucAAuAGcTsT 62 A22798GCuAUUGAUGUcAcAAuAATsT AD-14039 63 A22799 GAAcccAGGAAAuAcAuuGTsT 64A22800 cAAUGuAUUUCCUGGGUUCTsT AD-14040 65 A22801 AAcccAGGAAAuAcAuuGcTsT66 A22802 GcAAUGuAUUUCCUGGGUUTsT AD-14041 67 A22803GccAcAGuuAuuGucAuGGTsT 68 A22804 CcAUGAcAAuAACUGUGGCTsT AD-14042 69A22805 cGAuGuGAAGAAGGAAAcATsT 70 A22806 UGUUUCCUUCUUcAcAUCGTsT AD-1404371 A22807 GAuGuGAAGAAGGAAAcAGTsT 72 A22808 CUGUUUCCUUCUUcAcAUCTsTAD-14044 73 A22809 uGuGAAGAAGGAAAcAGGATsT 74 A22810UCCUGUUUCCUUCUUcAcATsT AD-14045 75 A22811 GuGAAGAAGGAAAcAGGAATsT 76A22812 UUCCUGUUUCCUUCUUcACTsT AD-14046 77 A22813 GuuAuGuuGAcAuucuuccTsT78 A22814 GGAAGAAUGUcAAcAuAACTsT AD-14047 79 A22815ccAcAGuuAuuGucAuGGuTsT 80 A22816 ACcAUGAcAAuAACUGUGGTsT AD-14048 81A22817 AAGGAAAcAGGAAcAAGuGTsT 82 A22818 cACUUGUUCCUGUUUCCUUTsT AD-1404983 A22819 GAAGAAGGAAAcAGGAAcATsT 84 A22820 uGUUCCuGUUUCCUUCUUCTsTAD-14050 85 A22821 uGGcuGAAuuucAGAGcAuTsT 86 A22822AUGCUCUGAAAUUcAGCcATsT AD-14051 87 A22823 GuuGAAAGGGAuGAuGAAATsT 88A22824 UUUcAUcAUCCCUUUcAACTsT AD-14052 89 A22825 cuGGcuGAAuuucAGAGcATsT90 A22826 UGCUCUGAAAUUcAGCcAGTsT AD-14053 91 A22827uucuGAuuAuuGuGAcAucTsT 92 A22828 GAUGUcAcAAuAAUcAGAATsT AD-14054 93A22829 AGAAGGAAAcAGGAAcAAGTsT 94 A22830 CUuGUUCCuGUUUCCUUCUTsT AD-1405595 A22831 uuucuGAuuAuuGuGAcAuTsT 96 A22832 AUGUcAcAAuAAUcAGAAATsT

TABLE 3 Efficacy of duplex dsRNAs IC80 Duplex Percent IC20 (nM) IC50(nM) (nM) IC20 (nM) IC50 (nM) IC80 (nM) Name Inhibition^(a) SD^(b)(FACS) (FACS) (FACS) (bDNA) (bDNA) (bDNA) AD-14008 86%  1% 0.8895931#N/A #N/A 0.03996853 0.2361971 #N/A AD-14009 34%  2% AD-14010 38%  3%AD-14011 11%  4% AD-14012 62%  5% AD-14013 60%  1% AD-14014 10%  8%AD-14015 71%  3% AD-14016 16% 10% AD-14017 46%  6% AD-14018 84%  0%0.01773181 0.12385771 #N/A AD-14019 50%  6% AD-14020 75%  3% AD-1402183%  5% 0.05351334 0.28749645 #N/A 0.00767305 0.06871733 3.50954466AD-14022 18%  9% AD-14023 82%  3% 0.16806395 1.15022087 #N/A 0.018542890.18153564 42.4598925 AD-14024 45%  8% AD-14025 72%  3% AD-14026 68%  4%AD-14027 20%  2% AD-14028 79%  3% 0.04248737 0.28602921 #N/A AD-1402954%  1% AD-14030 74%  3% AD-14031 79%  4% 0.6082823 1.9630781 #N/AAD-14032 61% 10% AD-14033 23%  6% AD-14034 55%  4% AD-14035 25%  0%AD-14036 63%  2% AD-14037 78%  7% AD-14038 12%  1% AD-14039 33%  5%AD-14040 17%  3% AD-14041 32%  0% AD-14042 78%  4% 0.00990567 0.08334966#N/A AD-14043 44%  5% AD-14044 59%  6% AD-14045 58%  4% AD-14046 50%  5%AD-14047 65%  2% AD-14048 12%  4% AD-14049 72%  2% AD-14050 52%  9%AD-14051 64%  3% AD-14052 89%  4% 0.06652968 0.30445773 #N/A 0.005431530.04500437 1.02650598 AD-14053 78%  3% 0.01679891 0.14666108 #N/AAD-14054 70%  8% AD-14055 26%  3% ^(a)Percent inhibition of CD45expression (relative to irrelevant control siRNA-treated cells; mean ofthree screens); by bDNA assay, 50 nM in P388D1. ^(b)Standard Deviation(mean of three screens)

TABLE 4 Exemplary unmodified dsRNAs targeting CD45. position of SenseAntisense 5′ base on SEQ strand sequence SEQ strand sequence transcriptID (5′ to 3′) Double ID (5′ to 3′) Double NM_002838.2 NO:overhang Design NO: overhang design 2208 147  GAAUAAAAACCGUUAUGUU 148AACAUAACGGUUUUUAUUC 2215 149  AACCGUUAUGUUGACAUUC 150GAAUGUCAACAUAACGGUU 1929 151  UCUGAUUAUUGUGACAUCA 152UGAUGUCACAAUAAUCAGA 2212 153  AAAAACCGUUAUGUUGACA 154UGUCAACAUAACGGUUUUU 2328 155  ACCCAGGAAAUACAUUGCU 156AGCAAUGUAUUUCCUGGGU 2206 157  CAGAAUAAAAACCGUUAUG 158CAUAACGGUUUUUAUUCUG 2207 159  AGAAUAAAAACCGUUAUGU 160ACAUAACGGUUUUUAUUCU 2411 161  CAGUUAUUGUCAUGGUCAC 162GUGACCAUGACAAUAACUG 2209 163  AAUAAAAACCGUUAUGUUG 164CAACAUAACGGUUUUUAUU 2331 165  CAGGAAAUACAUUGCUGCA 166UGCAGCAAUGUAUUUCCUG 2214 167  AAACCGUUAUGUUGACAUU 168AAUGUCAACAUAACGGUUU 2213 169  AAAACCGUUAUGUUGACAU 170AUGUCAACAUAACGGUUUU 2217 171  CCGUUAUGUUGACAUUCUU 172AAGAAUGUCAACAUAACGG 2216 173  ACCGUUAUGUUGACAUUCU 174AGAAUGUCAACAUAACGGU 2211 175  UAAAAACCGUUAUGUUGAC 176GUCAACAUAACGGUUUUUA 2218 177  CGUUAUGUUGACAUUCUUC 178GAAGAAUGUCAACAUAACG 2410 179  ACAGUUAUUGUCAUGGUCA 180UGACCAUGACAAUAACUGU 1931 181  UGAUUAUUGUGACAUCAAU 182AUUGAUGUCACAAUAAUCA 1932 183  GAUUAUUGUGACAUCAAUA 184UAUUGAUGUCACAAUAAUC 2332 185  AGGAAAUACAUUGCUGCAC 186GUGCAGCAAUGUAUUUCCU 2030 187  UUGAAAGGGAUGAUGAAAA 188UUUUCAUCAUCCCUUUCAA 2210 189  AUAAAAACCGUUAUGUUGA 190UCAACAUAACGGUUUUUAU 1930 191  CUGAUUAUUGUGACAUCAA 192UUGAUGUCACAAUAAUCAG 2329 193  CCCAGGAAAUACAUUGCUG 194CAGCAAUGUAUUUCCUGGG 2333 195 GGAAAUACAUUGCUGCACA 196 UGUGCAGCAAUGUAUUUCC2409 197 CACAGUUAUUGUCAUGGUC 198 GACCAUGACAAUAACUGUG 2438 199AAGAAGGAAACAGGAACAA 200 UUGUUCCUGUUUCCUUCUU 1933 201 AUUAUUGUGACAUCAAUAG202 CUAUUGAUGUCACAAUAAU 2330 203 CCAGGAAAUACAUUGCUGC 204GCAGCAAUGUAUUUCCUGG 2433 205 AUGUGAAGAAGGAAACAGG 206 CCUGUUUCCUUCUUCACAU1934 207 UUAUUGUGACAUCAAUAGC 208 GCUAUUGAUGUCACAAUAA 2326 209GAACCCAGGAAAUACAUUG 210 CAAUGUAUUUCCUGGGUUC 2327 211 AACCCAGGAAAUACAUUGC212 GCAAUGUAUUUCCUGGGUU 2407 213 GCCACAGUUAUUGUCAUGG 214CCAUGACAAUAACUGUGGC 2431 215 CGAUGUGAAGAAGGAAACA 216 UGUUUCCUUCUUCACAUCG2432 217 GAUGUGAAGAAGGAAACAG 218 CUGUUUCCUUCUUCACAUC 2434 219UGUGAAGAAGGAAACAGGA 220 UCCUGUUUCCUUCUUCACA 2435 221 GUGAAGAAGGAAACAGGAA222 UUCCUGUUUCCUUCUUCAC 2219 223 GUUAUGUUGACAUUCUUCC 224GGAAGAAUGUCAACAUAAC 2408 225 CCACAGUUAUUGUCAUGGU 226 ACCAUGACAAUAACUGUGG2441 227 AAGGAAACAGGAACAAGUG 228 CACUUGUUCCUGUUUCCUU 2437 229GAAGAAGGAAACAGGAACA 230 UGUUCCUGUUUCCUUCUUC 2135 231 UGGCUGAAUUUCAGAGCAU232 AUGCUCUGAAAUUCAGCCA 2029 233 GUUGAAAGGGAUGAUGAAA 234UUUCAUCAUCCCUUUCAAC 2134 235 CUGGCUGAAUUUCAGAGCA 236 UGCUCUGAAAUUCAGCCAG1928 237 UUCUGAUUAUUGUGACAUC 238 GAUGUCACAAUAAUCAGAA 2439 239AGAAGGAAACAGGAACAAG 240 CUUGUUCCUGUUUCCUUCU 1927 241 UUUCUGAUUAUUGUGACAU242 AUGUCACAAUAAUCAGAAAExemplary dsRNAs having NN-dinucleotide overhangs and targeting CD45.position of Sense Antisense 5′ base on SEQ strand sequence SEQstrand sequence transcript ID (5′ to 3′) Double ID (5′ to 3′) DoubleNM_002838.2 NO: overhang Design NO: overhang design 2208 243GAAUAAAAACCGUUAUGUUNN 244 AACAUAACGGUUUUUAUUCNN 2215 245AACCGUUAUGUUGACAUUCNN 246 GAAUGUCAACAUAACGGUUNN 1929 247UCUGAUUAUUGUGACAUCANN 248 UGAUGUCACAAUAAUCAGANN 2212 249AAAAACCGUUAUGUUGACANN 250 UGUCAACAUAACGGUUUUUNN 2328 251ACCCAGGAAAUACAUUGCUNN 252 AGCAAUGUAUUUCCUGGGUNN 2206 253CAGAAUAAAAACCGUUAUGNN 254 CAUAACGGUUUUUAUUCUGNN 2207 255AGAAUAAAAACCGUUAUGUNN 256 ACAUAACGGUUUUUAUUCUNN 2411 257CAGUUAUUGUCAUGGUCACNN 258 GUGACCAUGACAAUAACUGNN 2209 259AAUAAAAACCGUUAUGUUGNN 260 CAACAUAACGGUUUUUAUUNN 2331 261CAGGAAAUACAUUGCUGCANN 262 UGCAGCAAUGUAUUUCCUGNN 2214 263AAACCGUUAUGUUGACAUUNN 264 AAUGUCAACAUAACGGUUUNN 2213 265AAAACCGUUAUGUUGACAUNN 266 AUGUCAACAUAACGGUUUUNN 2217 267CCGUUAUGUUGACAUUCUUNN 268 AAGAAUGUCAACAUAACGGNN 2216 269ACCGUUAUGUUGACAUUCUNN 270 AGAAUGUCAACAUAACGGUNN 2211 271UAAAAACCGUUAUGUUGACNN 272 GUCAACAUAACGGUUUUUANN 2218 273CGUUAUGUUGACAUUCUUCNN 274 GAAGAAUGUCAACAUAACGNN 2410 275ACAGUUAUUGUCAUGGUCANN 276 UGACCAUGACAAUAACUGUNN 1931 277UGAUUAUUGUGACAUCAAUNN 278 AUUGAUGUCACAAUAAUCANN 1932 279GAUUAUUGUGACAUCAAUANN 280 UAUUGAUGUCACAAUAAUCNN 2332 281AGGAAAUACAUUGCUGCACNN 282 GUGCAGCAAUGUAUUUCCUNN 2030 283UUGAAAGGGAUGAUGAAAANN 284 UUUUCAUCAUCCCUUUCAANN 2210 285AUAAAAACCGUUAUGUUGANN 286 UCAACAUAACGGUUUUUAUNN 1930 287CUGAUUAUUGUGACAUCAANN 288 UUGAUGUCACAAUAAUCAGNN 2329 289CCCAGGAAAUACAUUGCUGNN 290 CAGCAAUGUAUUUCCUGGGNN 2333 291GGAAAUACAUUGCUGCACANN 292 UGUGCAGCAAUGUAUUUCCNN 2409 293CACAGUUAUUGUCAUGGUCNN 294 GACCAUGACAAUAACUGUGNN 2438 295AAGAAGGAAACAGGAACAANN 296 UUGUUCCUGUUUCCUUCUUNN 1933 297AUUAUUGUGACAUCAAUAGNN 298 CUAUUGAUGUCACAAUAAUNN 2330 299CCAGGAAAUACAUUGCUGCNN 300 GCAGCAAUGUAUUUCCUGGNN 2433 301AUGUGAAGAAGGAAACAGGNN 302 CCUGUUUCCUUCUUCACAUNN 1934 303UUAUUGUGACAUCAAUAGCNN 304 GCUAUUGAUGUCACAAUAANN 2326 305GAACCCAGGAAAUACAUUGNN 306 CAAUGUAUUUCCUGGGUUCNN 2327 307AACCCAGGAAAUACAUUGCNN 308 GCAAUGUAUUUCCUGGGUUNN 2407 309GCCACAGUUAUUGUCAUGGNN 310 CCAUGACAAUAACUGUGGCNN 2431 311CGAUGUGAAGAAGGAAACANN 312 UGUUUCCUUCUUCACAUCGNN 2432 313GAUGUGAAGAAGGAAACAGNN 314 CUGUUUCCUUCUUCACAUCNN 2434 315UGUGAAGAAGGAAACAGGANN 316 UCCUGUUUCCUUCUUCACANN 2435 317GUGAAGAAGGAAACAGGAANN 318 UUCCUGUUUCCUUCUUCACNN 2219 319GUUAUGUUGACAUUCUUCCNN 320 GGAAGAAUGUCAACAUAACNN 2408 321CCACAGUUAUUGUCAUGGUNN 322 ACCAUGACAAUAACUGUGGNN 2441 323AAGGAAACAGGAACAAGUGNN 324 CACUUGUUCCUGUUUCCUUNN 2437 325GAAGAAGGAAACAGGAACANN 326 UGUUCCUGUUUCCUUCUUCNN 2135 327UGGCUGAAUUUCAGAGCAUNN 328 AUGCUCUGAAAUUCAGCCANN 2029 329GUUGAAAGGGAUGAUGAAANN 330 UUUCAUCAUCCCUUUCAACNN 2134 331CUGGCUGAAUUUCAGAGCANN 332 UGCUCUGAAAUUCAGCCAGNN 1928 333UUCUGAUUAUUGUGACAUCNN 334 GAUGUCACAAUAAUCAGAANN 2439 335AGAAGGAAACAGGAACAAGNN 336 CUUGUUCCUGUUUCCUUCUNN 1927 337UUUCUGAUUAUUGUGACAUNN 338 AUGUCACAAUAAUCAGAAANN

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the invention, suitable methods and materials aredescribed below. All publications, patent applications, patents, andother references mentioned herein are incorporated by reference in theirentirety. In case of conflict, the present specification, includingdefinitions, will control. In addition, the materials, methods, andexamples are illustrative only and not intended to be limiting.

EXAMPLES Example 1 In Silico Selection of siRNAs Targeting CD45

siRNA design was carried out to identify siRNAs targeting CD45 (alsoknown PTPRC, B220, CD45, GP180, LCA, LY5, and T200). Human mRNAsequences to CD45, RefSeq ID number: NM_(—)002838.3, NM_(—)080921.2,NM_(—)080922.2, NM_(—)080923.2, Y00062.1, Y00638.1, BC014239.2,BC017863.1, BC031525.1, BC121086.1, BC121087.1, BC127656.1, BC127657.1,AY429565.1, AY567999.1, AK130573.1, DA670254.1, DA948670.1, AY429566.1,and CR621867.1 were used. Mouse mRNA sequences to CD45, RefSeq IDnumber: NM_(—)011210.2, AK054056.1, AK088215.1, AK154893.1, AK171802.1,BC028512.1, EF101553.1, L36091.1, M11934.1, M14342.1, M14343.1,M15174.1, M17320.1, and M92933.1 were used. Rhesus monkey mRNA sequenceto CD45, RefSeqID number; XR_(—)012672.1 was also used.

siRNA duplexes cross-reactive to human, mouse and rhesus CD45transcripts were designed. Forty-eight duplexes were synthesized andscreened as outlined in Tables 1-3.

The sequences for human, mouse and partial rhesus CD45 mRNAs weredownloaded from NCBI Nucleotide database and the human sequence (HumanCD45: NM_(—)002838.3, 5330 bp) was further used as reference sequence.

For identification of further rhesus CD45 sequences, a blast search withthe human reference sequence was conducted at NCBI against the rhesusreference genome. The downloaded rhesus sequence and the hit regions inthe blast hit were assembled to a rhesus consensus sequence with 91%identity to human CD45 reference sequence over the full-length.

All conserved 19mers were extracted from the human mRNA referencesequence, resulting in the pool of candidate target sites correspondingto 5312 (sense strand) sequences. Human-mouse-rhesus cross-reactivitywas defined as a prerequisite for in silico selection of siRNAs out ofthis candidate pool. As no conserved regions are present in all humanand/or mouse variants, the criterion for selection was relaxed tocross-reactivity to most relevant human and mouse CD45 sequences, whichwe assumed to be RefSeq sequences as well as other mRNAs for whichprotein expression has been described.

To determine cross-reactivity to human and mouse CD45 variants andrhesus-reactive siRNAs, the presence of each candidate siRNA target sitewas searched in the sequences.

Further, the predicted specificity of the siRNA was used as criterionfor selection out of the pool of human-mouse-rhesus cross-reactivesiRNAs, manifested by targeting human CD45 mRNA sequences, but not otherhuman mRNAs.

The specificity of an siRNA can be expressed via its potential to targetother genes, which are referred to as “off-target genes.” For predictingthe off-target potential of an siRNA, the following assumptions weremade: (1) off-target potential of a strand can be deduced from thenumber and distribution of mismatches to an off-target; (2) the mostrelevant off-target, that is the gene predicted to have the highestprobability to be silenced due to tolerance of mismatches, determinesthe off-target potential of the strand; (3) positions 2 to 9 (counting5′ to 3′) of a strand (seed region) may contribute more to off-targetpotential than the rest of the sequence (that is non-seed and cleavagesite region); (4) positions 10 and 11 (counting 5′ to 3′) of a strand(cleavage site region) may contribute more to off-target potential thannon-seed region (that is positions 12 to 18, counting 5′ to 3′); (5)positions 1 and 19 of each strand are not relevant for off-targetinteractions; (6) off-target potential can be expressed by theoff-target score of the most relevant off-target, calculated based onnumber and position of mismatches of the strand to the most homologousregion in the off-target gene considering assumptions 3 to 5; and (7)off-target potential of antisense and sense strand will be relevant,whereas potential abortion of sense strand activity by internalmodifications introduced is likely.

SiRNAs with low off-target potential were defined as preferable andassumed to be more specific.

In order to identify human CD45-specific siRNAs, all other humantranscripts that were considered potential off-targets, were searchedfor potential target regions for human-mouse-rhesus cross-reactive 19mersense strand sequences as well as complementary antisense strands. Forthis, the fastA algorithm was used to determine the most homologous hitregion in each sequence of the human RefSeq database, which we assume torepresent the comprehensive human transcriptome.

FastA output files were analyzed further by a Perl script to rank allpotential off-targets according to assumptions 3′ to 5′, and thus toidentify the most relevant off-target gene and its off-target score.

The script extracted the following off-target properties for each 19merinput sequence and each off-target gene to calculate the off-targetscore:

Number of mismatches in non-seed region, number of mismatches in seedregion, and number of mismatches in cleavage site region.

The off-target score was calculated by considering assumptions 3 to 5 asfollows:

Off-target  score = number  of  seed  mismatches * 10 + number  of  cleavage  site  mismatches * 1.2 + number  of  non-seed  mismatches * 1

The most relevant off-target gene for each 19mer sequence was defined asthe gene with the lowest off-target score. Accordingly, the lowestoff-target score was defined as representative for the off-targetpotential of a strand.

For the siRNA set in Table 2, cross-reactivity to rhesus, all human andmouse RefSeq sequences as well as to most variants with describedprotein was defined as prerequisite for selection. Further criterion wasan off-target score of 1 or more for the antisense strand, whereas allsequences containing 4 or more consecutive G's (poly-G sequences) wereexcluded. 48 human-mouse-rhesus cross-reactive sequences that do notpossess most-relevant off-targets predicted to be expressed in immunecells were selected (Tables 1 and 2).

Table 1 shows CD45 target sequences, Table 2 shows CD45 siRNAs, andTable 3 shows the IC values for the siRNAs tested in a dose responseassay. Table 4 shows exemplary CD45 siRNAs that are not modified, andTable 5 shows exemplary CD45 siRNAs having dinucleotide overhangs.

Nucleic acid sequences are represented below using standardnomenclature, and specifically the abbreviations of Table 6.

TABLE 6 Abbreviations of nucleotide monomers used in nucleic acidsequence representation. It will be understood that these monomers, whenpresent in an oligonucleotide, are mutually linked by5′-3′-phosphodiester bonds. Abbreviation^(a) Nucleotide(s) A Adenosine Ccytidine G guanosine T thymidine U uridine N any nucleotide (G, A, C, U,T) a 2′-O-methyladenosine c 2′-O-methylcytidine g 2′-O-methylguanosine u2′-O-methyluridine dT 2′-deoxythymidine s a phosphorothioate linkage

Example 2 Screening Assay

48 human-mouse-rhesus cross-reactive siRNAs were first screened at 50 nMsingle dose (three independent screens, each done in quadruplicate) bybDNA assay in P388D1 cells. For the most potent siRNAs, dose responsewas performed by bDNA analysis in P388D1 to identify the IC20, IC50 andIC80 dose that lowers the level of CD45 transcript by 20, 50 and 80%,respectively. siRNAs with the best IC values in the bDNA assay werescreened by flow cytometry to identify the IC20, IC50 and IC80 doserequired to lower the amount of CD45 protein by 20, 50 and 80%,respectively.

Cell Line.

A P388D1 mouse macrophage cell line was obtained from the American TypeCulture Collection (ATCC, Rockville Md., USA; ATCC # TIB-63) and grownin DMEM containing 10% heat-inactivated fetal bovine serum (FBS), 4 mML-glutamine, 1.5 g/L sodium bicarbonate at 37° C. under a 5% CO2/95% airatmosphere at 37° C.

Cell Culture, siRNA Transfection.

P388D1 cells were plated in 24-well plates at 8×10⁴ cells per well in0.4 ml growth medium a day before transfection. P388D1 cells were 80%confluent the day of siRNA transfection. Before transfection, cells arefed with 0.25 ml growth medium.

Prior to adding to cells, 1.5 ml (50 μl per well) Optimem I (Invitrogen)and 90 μl (3 μl per well) Lipofectamin 2000 (Invitrogen), the amountsufficient for transfection of one 24 well plate, were combined in a 2ml Sarstedt tube and incubated for 10-15 minutes at room temperature.The appropriate amount of siRNA dissolved in transfection buffer is thenadded to the Optimem/lipofectamine 2000 mixture to give the desiredfinal concentration, mixed, and incubated an additional 15-25 minutes atroom temperature. 50 μl of the siRNA/reagent complex was then addeddropwise to each well as dictated by the experimental design. Plateswere then gently rocked to ensure complete mixing and incubated at 37°C. at 5% CO₂/95% air for 48 hours. The cells were lysed and CD45 mRNAtranscript was quantitated in relation to a house-keeping genetranscript by branched DNA (bDNA) assay. CD45 protein expression levelwas determined by flow cytometry, for this assay cells were harvested bypipetting without lysing.

siRNA to CD45 Silenced CD45 in Mouse Macrophages In Vivo

C57B1/6J mice (Jackson Labs) were injected intraperitoneally with 1 mLof 4% Brewers thioglycollate medium (Difco) 3 days prior to injecting 10mg/kg of 98N12-5 formulated siCD45 or siGFP i.p. (4 mice per group). Thethioglycollate acted as a sterile inflammation stimulus. Peritoneallavage was collected 4 days later and stained with fluorophoreconjugated antibodies to CD11b, Gr1 and CD45 (BD Biosciences). Flowcytometry samples were run on the LSRII flow cytometer (BD Biosciences)and FlowJo software (Treestar) was used to identify the CD11b^(high)Gr1^(low) macrophage population and quantify CD45 expression. A 65%reduction of CD45 protein expression was observed in the peritonealmacrophage population when treated with the formulated CD45 siRNA (FIG.1). Two independent dose response experiments were conducted examiningthe effect of 0.6-15.0 mg/kg administration of 98N12-5 formulated siCD45or siGFP i.p. (2-3 mice per group). These experiments were conductedidentically to those described above and demonstrated effective specificin vivo silencing of CD45 protein expression at all concentrationstested when treated with the formulated CD45 siRNA (FIG. 1, top panel).The 98N12-5 formulation is a lipidoid synthesized by addition ofacrylamides or acrylates to amines.

The sequences for the sense and antisense strands of the CD45 siRNA areas follows.

sense 5′-cuGGcuGAAuuucAGAGcATsT-3′ (SEQ ID NO: 89 single strand #A22825) antisense 5′-UGCUCUGAAAUUcAGCcAGTsT-3′(SEQ ID NO: 90 single strand #_A22826)The siGFP sequences are as follows: sense (SEQ ID NO: 145)5′-CcAcAuGAAGcAGcACGACusU-3′ (single strand # AL4545) antisense(SEQ ID NO: 146) 5′-AAGUCGUGCUGCUUCAUGUGgsusC-3′ (single strand #AL4381)

2′-O-Me modified nucleotides are in lower case, and phosphorothioatelinkages are represented by an “s”. siRNAs were generated by annealingequimolar amounts of complementary sense and antisense strands.

All procedures used in animal studies were approved by the InstitutionalAnimal Care and Use Committee (IACUC) and were consistent with local,state, and federal regulations as applicable.

Lipidoid-based siRNA formulations included lipidoid, cholesterol,poly(ethylene glycol)-lipid (PEG-lipid), and siRNA. Formulations wereprepared using a protocol similar to that described by Semple andcolleagues (Maurer et al. Biophys. J. 80:2310-2326, 2001; Semple et al.,Biochim. Biophys. Acta 1510:152-166, 2001). Stock solutions of98N12-5(1).4HCl MW 1489, mPEG2000-Ceramide C16 (Avanti Polar Lipids) MW2634 or mPEG2000-DMG MW 2660, and cholesterol MW 387 (Sigma-Aldrich)were prepared in ethanol and mixed to yield a molar ratio of 42:10:48.Mixed lipids were added to 125 mM sodium acetate buffer pH 5.2 to yielda solution containing 35% ethanol, resulting in spontaneous formation ofempty lipidoid nanoparticles. Resulting nanoparticles were extrudedthrough a 0.08μ membrane (2 passes). siRNA in 35% ethanol and 50 mMsodium acetate pH 5.2 was added to the nanoparticles at 1:7.5 (wt:wt)siRNA:total lipids and incubated at 37° C. for 30 min. Ethanol removaland buffer exchange of siRNA-containing lipidoid nanoparticles wasachieved by tangential flow filtration against phosphate buffered salineusing a 100,000 MWCO membrane. Finally, the formulation was filteredthrough a 0.2μ sterile filter. Particle size was determined using aMalvern Zetasizer NanoZS (Malvern, UK). siRNA content was determined byUV absorption at 260 nm and siRNA entrapment efficiency was determinedby Ribogreen assay 32. Resulting particles had a mean particle diameterof approximately 50 nm, with peak width of 20 nm, and siRNA entrapmentefficiency of >95%. See also PCT/US2007/080331.

Example 3 Bone Marrow-Derived Macrophage Transfection

Murine bone marrow derived macrophages were cultured according tostandard protocol (Cunnick et al., J. Immunol. Methods 311:96-105,2006). Cells were cultured in 12-well dishes for five days in thepresence of 8 ng/mL of M-CSF. The optimal siRNA to lipidoid ratio wasdetermined for each lipidoid (a ratio of either 5 or 10 wt:wt was used).Mixtures of irrelevant control siRNA or siCD45 (SEQ ID NOs:89 and 90,above) with lipidoids were prepared as described above. siRNA-lipidoidmixtures were added to macrophage cultures at the desired concentrationsfor 6 hours. Media was exchanged and GFP expression was analyzed by flowcytometry five days later. The formulated CD45 siRNA were shown toeffectively silence primary murine bone marrow-derived macrophages with65% protein reduction (FIG. 2)

Example 4 CD45 as a Cellular Protein Target that Regulates Infectionfrom a Broad Range of Pathogens

CD45 mitigates viral and bacterial infections. Genetically-modified micewith reduced expression level of CD45 were protected from B. anthracis,influenza, and Ebola (see FIG. 3). Given the ability of formulated CD45siRNA to inhibit CD45 expression in vitro and in vivo by up to 65%, itis notable that significant protective effects against lethal Ebolachallenge were seen in genetically-modified mice when CD45 expressionwas reduced by 11-65% (FIG. 3).

Example 5 CD45 siRNA Silenced CD45 Expression in Human Cells In Vitro

The human acute myelogenous leukemia cell line, KG-1, was culturedaccording to standard protocol. Cells were plated into 96 wells and thenuntreated or treated with CD45 siRNA-lipidoid mixtures (as outlined forFIGS. 1 and 2) at the desired concentrations for 6 hours. Media wasexchanged and CD45 expression was analyzed by flow cytometry four dayslater. The liposomally formulated CD45 siRNA were shown to effectivelysilence in human cells in vitro (FIG. 4). These results are consistentwith the fact that the CD45 siRNA has 100% sequence identity to humanand rodent CD45 and was shown to be active in reducing murine CD45 invitro and in vivo (Table 3; FIGS. 1 and 2).

Example 6 dsRNA Synthesis

Source of Reagents

Where the source of a reagent is not specifically given herein, suchreagent may be obtained from any supplier of reagents for molecularbiology at a quality/purity standard for application in molecularbiology.

siRNA synthesis

Single-stranded RNAs were produced by solid phase synthesis on a scaleof 1 μmole using an Expedite 8909 synthesizer (Applied Biosystems,Applera Deutschland GmbH, Darmstadt, Germany) and controlled pore glass(CPG, 500 Å, Proligo Biochemie GmbH, Hamburg, Germany) as solid support.RNA and RNA containing 2′-O-methyl nucleotides were generated by solidphase synthesis employing the corresponding phosphoramidites and2′-O-methyl phosphoramidites, respectively (Proligo Biochemie GmbH,Hamburg, Germany). These building blocks were incorporated at selectedsites within the sequence of the oligoribonucleotide chain usingstandard nucleoside phosphoramidite chemistry such as described inCurrent protocols in nucleic acid chemistry, Beaucage, S. L. et al.(Edrs.), John Wiley & Sons, Inc., New York, N.Y., USA. Phosphorothioatelinkages were introduced by replacement of the iodine oxidizer solutionwith a solution of the Beaucage reagent (Chruachem Ltd, Glasgow, UK) inacetonitrile (1%). Further ancillary reagents were obtained fromMallinckrodt Baker (Griesheim, Germany).

Deprotection and purification of the crude oligoribonucleotides by anionexchange HPLC were carried out according to established procedures.Yields and concentrations were determined by UV absorption of a solutionof the respective RNA at a wavelength of 260 nm using a spectralphotometer (DU 640B, Beckman Coulter GmbH, Unterschleiβheim, Germany).Double stranded RNA was generated by mixing an equimolar solution ofcomplementary strands in annealing buffer (20 mM sodium phosphate, pH6.8; 100 mM sodium chloride), heated in a water bath at 85-90° C. for 3minutes and cooled to room temperature over a period of 3-4 hours. Theannealed RNA solution was stored at −20° C. until use.

For the synthesis of 3′-cholesterol-conjugated siRNAs (herein referredto as -Chol-3), an appropriately modified solid support is used for RNAsynthesis. The modified solid support is prepared as follows:

Diethyl-2-azabutane-1,4-dicarboxylate AA

A 4.7 M aqueous solution of sodium hydroxide (50 mL) is added into astirred, ice-cooled solution of ethyl glycinate hydrochloride (32.19 g,0.23 mole) in water (50 mL). Then, ethyl acrylate (23.1 g, 0.23 mole) isadded and the mixture is stirred at room temperature until completion ofthe reaction is ascertained by TLC. After 19 h the solution ispartitioned with dichloromethane (3×100 mL). The organic layer is driedwith anhydrous sodium sulfate, filtered and evaporated. The residue isdistilled to afford AA (28.8 g, 61%).

3-{Ethoxycarbonylmethyl-[6-(9H-fluoren-9-ylmethoxycarbonyl-amino)-hexanoyl]-amino}-propionicacid ethyl ester AB

Fmoc-6-amino-hexanoic acid (9.12 g, 25.83 mmol) is dissolved indichloromethane (50 mL) and cooled with ice. Diisopropylcarbodiimde(3.25 g, 3.99 mL, 25.83 mmol) is added to the solution at 0° C. It isthen followed by the addition of Diethyl-azabutane-1,4-dicarboxylate (5g, 24.6 mmol) and dimethylamino pyridine (0.305 g, 2.5 mmol). Thesolution is brought to room temperature and stirred further for 6 h.Completion of the reaction is ascertained by TLC. The reaction mixtureis concentrated under vacuum and ethyl acetate is added to precipitatediisopropyl urea. The suspension is filtered. The filtrate is washedwith 5% aqueous hydrochloric acid, 5% sodium bicarbonate and water. Thecombined organic layer is dried over sodium sulfate and concentrated togive the crude product which is purified by column chromatography (50%EtOAC/Hexanes) to yield 11.87 g (88%) of AB.

3-[(6-Amino-hexanoyl)-ethoxycarbonylmethyl-amino]-propionic acid ethylester AC

3-{Ethoxycarbonylmethyl-[6-(9H-fluoren-9-ylmethoxycarbonylamino)-hexanoyl]-amino}-propionicacid ethyl ester AB (11.5 g, 21.3 mmol) is dissolved in 20% piperidinein dimethylformamide at 0° C. The solution is continued stirring for 1h. The reaction mixture is concentrated under vacuum, water is added tothe residue, and the product is extracted with ethyl acetate. The crudeproduct is purified by conversion into its hydrochloride salt.

3-({6-[17-(1,5-Dimethyl-hexyl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yloxycarbonylamino]-hexanoyl}ethoxycarbonylmethyl-amino)-propionicacid ethyl ester AD

The hydrochloride salt of3-[(6-Amino-hexanoyl)-ethoxycarbonylmethyl-amino]-propionic acid ethylester AC (4.7 g, 14.8 mmol) is taken up in dichloromethane. Thesuspension is cooled to 0° C. on ice. To the suspensiondiisopropylethylamine (3.87 g, 5.2 mL, 30 mmol) is added. To theresulting solution cholesteryl chloroformate (6.675 g, 14.8 mmol) isadded. The reaction mixture is stirred overnight. The reaction mixtureis diluted with dichloromethane and ished with 10% hydrochloric acid.The product is purified by flash chromatography (10.3 g, 92%).

1-{6-[17-(1,5-Dimethyl-hexyl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yloxycarbonylamino]-hexanoyl}-4-oxo-pyrrolidine-3-carboxylicacid ethyl ester AE

Potassium t-butoxide (1.1 g, 9.8 mmol) is slurried in 30 mL of drytoluene. The mixture is cooled to 0° C. on ice and 5 g (6.6 mmol) ofdiester AD is added slowly with stirring within 20 mins. The temperatureis kept below 5° C. during the addition. The stirring is continued for30 mins at 0° C. and 1 mL of glacial acetic acid is added, immediatelyfollowed by 4 g of NaH₂PO₄.H₂O in 40 mL of water The resultant mixtureis extracted twice with 100 mL of dichloromethane each and the combinedorganic extracts are washed twice with 10 mL of phosphate buffer each,dried, and evaporated to dryness. The residue is dissolved in 60 mL oftoluene, cooled to 0° C. and extracted with three 50 mL portions of coldpH 9.5 carbonate buffer. The aqueous extracts are adjusted to pH 3 withphosphoric acid, and extracted with five 40 mL portions of chloroformwhich are combined, dried and evaporated to dryness. The residue ispurified by column chromatography using 25% ethylacetate/hexane toafford 1.9 g of b-ketoester (39%).

[6-(3-Hydroxy-4-hydroxymethyl-pyrrolidin-1-yl)-6-oxo-hexyl]-carbamicacid17-(1,5-dimethyl-hexyl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-ylester AF

Methanol (2 mL) is added dropwise over a period of 1 h to a refluxingmixture of b-ketoester AE (1.5 g, 2.2 mmol) and sodium borohydride(0.226 g, 6 mmol) in tetrahydrofuran (10 mL). Stirring is continued atreflux temperature for 1 h. After cooling to room temperature, 1 N HCl(12.5 mL) is added, the mixture is extracted with ethylacetate (3×40mL). The combined ethylacetate layer is dried over anhydrous sodiumsulfate and concentrated under vacuum to yield the product which ispurified by column chromatography (10% MeOH/CHCl₃) (89%).

(6-{3-[Bis-(4-methoxy-phenyl)-phenyl-methoxymethyl]-4-hydroxy-pyrrolidin-1-yl}-6-oxo-hexyl)-carbamicacid17-(1,5-dimethyl-hexyl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-ylester AG

Diol AF (1.25 gm 1.994 mmol) is dried by evaporating with pyridine (2×5mL) in vacuo. Anhydrous pyridine (10 mL) and4,4′-dimethoxytritylchloride (0.724 g, 2.13 mmol) are added withstirring. The reaction is carried out at room temperature overnight. Thereaction is quenched by the addition of methanol. The reaction mixtureis concentrated under vacuum and to the residue dichloromethane (50 mL)is added. The organic layer is washed with 1M aqueous sodiumbicarbonate. The organic layer is dried over anhydrous sodium sulfate,filtered and concentrated. The residual pyridine is removed byevaporating with toluene. The crude product is purified by columnchromatography (2% MeOH/Chloroform, Rf=0.5 in 5% MeOH/CHCl₃) (1.75 g,95%).

Succinic acidmono-(4-[bis-(4-methoxy-phenyl)-phenyl-methoxymethyl]-1-{6-[17-(1,5-dimethyl-hexyl)-10,13-dimethyl2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1Hcyclopenta[a]phenanthren-3-yloxycarbonylamino]-hexanoyl}-pyrrolidin-3-yl)esterAH

Compound AG (1.0 g, 1.05 mmol) is mixed with succinic anhydride (0.150g, 1.5 mmol) and DMAP (0.073 g, 0.6 mmol) and dried in a vacuum at 40°C. overnight. The mixture is dissolved in anhydrous dichloroethane (3mL), triethylamine (0.318 g, 0.440 mL, 3.15 mmol) is added and thesolution is stirred at room temperature under argon atmosphere for 16 h.It is then diluted with dichloromethane (40 mL) and washed with ice coldaqueous citric acid (5 wt %, 30 mL) and water (2×20 mL). The organicphase is dried over anhydrous sodium sulfate and concentrated todryness. The residue is used as such for the next step.

Cholesterol Derivatised CPG AI

Succinate AH (0.254 g, 0.242 mmol) is dissolved in a mixture ofdichloromethane/acetonitrile (3:2, 3 mL). To that solution DMAP (0.0296g, 0.242 mmol) in acetonitrile (1.25 mL),2,2′-Dithio-bis(5-nitropyridine) (0.075 g, 0.242 mmol) inacetonitrile/dichloroethane (3:1, 1.25 mL) are added successively. Tothe resulting solution triphenylphosphine (0.064 g, 0.242 mmol) inacetonitrile (0.6 ml) is added. The reaction mixture turned brightorange in color. The solution is agitated briefly using a wrist-actionshaker (5 mins). Long chain alkyl amine-CPG (LCAA-CPG) (1.5 g, 61 mM) isadded. The suspension is agitated for 2 h. The CPG is filtered through asintered funnel and washed with acetonitrile, dichloromethane and ethersuccessively. Unreacted amino groups are masked using aceticanhydride/pyridine. The achieved loading of the CPG is measured bytaking UV measurement (37 mM/g).

The synthesis of siRNAs bearing a 5′-12-dodecanoic acid bisdecylamidegroup (herein referred to as “5′-C32-”) or a 5′-cholesteryl derivativegroup (herein referred to as “5′-Chol-”) is performed as described in WO2004/065601, except that, for the cholesteryl derivative, the oxidationstep is performed using the Beaucage reagent in order to introduce aphosphorothioate linkage at the 5′-end of the nucleic acid oligomer.

Other embodiments are in the claims.

We claim:
 1. An isolated double-stranded ribonucleic acid (dsRNA),wherein said dsRNA comprises a sense strand and an antisense strand thatare substantially complementary to each other and are each 15-30nucleotides in length, and the antisense strand comprising a region ofcomplementarity that is substantially complementary to an mRNA encodinga CD45.
 2. The dsRNA of claim 1, wherein said dsRNA, upon contact with acell expressing said CD45, inhibits expression of said CD45 by at least20%.
 3. The dsRNA of claim 1, wherein said dsRNA comprises at least onemodified nucleotide.
 4. The dsRNA of claim 3, wherein said modifiednucleotide is chosen from the group consisting of: a 2′-O-methylmodified nucleotide, a nucleotide comprising a 5′-phosphorothioategroup, and a terminal nucleotide linked to a cholesteryl derivative ordodecanoic acid bisdecylamide group.
 5. The dsRNA of claim 3, whereinsaid modified nucleotide is chosen from the group consisting of: a2′-deoxy-2′-fluoro modified nucleotide, a 2′-deoxy-modified nucleotide,a locked nucleotide, an abasic nucleotide, 2′-amino-modified nucleotide,2′-alkyl-modified nucleotide, morpholino nucleotide, a phosphoramidate,and a non-natural base comprising nucleotide.
 6. The dsRNA of claim 1,wherein said region of complementarity is at least 15 nucleotides inlength.
 7. The dsRNA of claim 1, wherein said region of complementarityis 19-24 nucleotides in length.
 8. The dsRNA of claim 1, wherein saiddsRNA, upon contact with a cell expressing CD45, inhibits expression ofCD45 by at least 20% as measured in the P388D1 cell assay.
 9. The dsRNAof claim 1, wherein said region of complementarity is complementary toat least 15 contiguous nucleotides of one of SEQ ID NOS:97-144.
 10. Acell comprising the dsRNA of claim
 1. 11. A pharmaceutical compositioncomprising the dsRNA of claim 1, and a pharmaceutically acceptablecarrier.
 12. The pharmaceutical composition of claim 11, wherein saiddsRNA, upon contact with a cell expressing said CD45 gene, inhibitsexpression of said CD45 gene by at least 20%, as measured in a P388D1cell assay.
 13. A method for inhibiting expression of a CD45 gene in acell, the method comprising: (a) introducing into the cell the dsRNA ofclaim 1; and (b) maintaining the cell produced in step (a) for a timesufficient to obtain degradation of an mRNA transcript of the CD45 gene,thereby inhibiting expression of the CD45 gene in the cell.
 14. Themethod of claim 13, wherein the cell produced in step (a) is maintainedfor a time sufficient inhibit expression expression of the CD45 gene by20%.
 15. A method of treating or managing an autoimmune diseasecomprising administering to a patient in need of such treatment ormanagement a effective amount of the dsRNA of claim
 1. 16. The method ofclaim 15, wherein the autoimmune disease is Graves' disease or multiplesclerosis.
 17. A method of treating or managing a viral infectioncomprising administering to a patient in need of such treatment ormanagement a therapeutically effective amount of the dsRNA of claim 1.18. The method of claim 17, wherein the infection is caused by a virusof the group consisting of Ebola, influenza, anthrax, hepatitis B andhepatitis C.
 19. A vector for inhibiting the expression of a CD45 genein a cell, said vector comprising a regulatory sequence operably linkedto a nucleotide sequence that encodes at least one strand of the dsRNAof claim
 1. 20. A cell comprising the vector of claim 19.